Chimeric T cell receptor comprising carbonic anhydrase IX (G250) antibody

ABSTRACT

The invention provides scFv antibodies and monoclonal antibodies that bind to and decrease an activity of Carbonic Anhydrase IX (G250). Also provided are methods of treating and/or preventing cancer, such as renal clear cell cancer. Also provided are methods of identifying a carbonic anhydrase IX (G250) protein. The invention additionally provides methods of modifying immune effector cells, and the immune effector cells modified thereby.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/095,773, filed Nov. 3, 2008, which entered the national stage under35 U.S.C. 371 and corresponds to International Application No.PCT/US2006/046350, filed Dec. 4, 2006, which claims priority to, and thebenefit of, U.S. Provisional Patent Application No. 60/742,149, filedDec. 2, 2005. The contents of each of these applications areincorporated herein by reference in their entireties.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with United States Government support undergrant number DK072282 awarded by The National Institutes of Health. Thegovernment may have certain rights in the invention.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “DFCI-040_C01US322270-2192_ST25.txt”, which was created on Mar. 7, 2016 and is 72 KB insize, are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates generally to anti-carbonic anhydrase IX (G250)antibodies as well as to methods for use thereof.

BACKGROUND

Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies andthere are approximately 32,000 new cases diagnosed each year in theUnited States. RCC is resistant to virtually all conventional modes oftreatment, such as radiotherapy and chemotherapy, reinforcing the urgentneed for new therapies. RCC is a clinicopathologically heterogeneousdisease, traditionally subdivided into clear cell, granular cell,papillary, chromophobe, spindle cell, cystic, and collecting ductcarcinoma subtypes based on morphological features according to the WHOInternational Histological Classification of Kidney Tumors (Mostfi,1998). Clear cell RCC is the most common adult renal neoplasm,representing 70% of all renal neoplasms, and is thought to originate inthe proximal tubules. Clear cell RCC is mostly sporadic, unilateral, andunifocal. The main genetic alterations of clear cell RCC have beenidentified to be chromosome 3 alterations and Von Hippel-Landau (VHL)gene mutations (Walsh, 2003). pVHL is part of a novel multiproteinubiquitin ligase complex, termed VBC (VHL/Elongin B/Elongin C), thatrecruits important cellular proteins for rapid degradation by theubiquitin-proteasome proteolysis system. Among the cellular proteinsthat bind to VHL and are normally degraded under normoxic conditions ishypoxia-inducible transcription factor (HIF1). HIF1 is considered to bea master regulator gene that integrates pathways regulatingphysiological responses to acute and chronic hypoxia. HIF1 controls theexpression of several dozen target genes, including those involved inenergy metabolism (glucose transporters, glycolytic enzymes),angiogenesis [vascular endothelial growth factor (VEGF) and VEGFR-1],and surface transmembrane carbonic anhydrases (CAs) (Hanahan, 1996;Ivanov, 1998; Maxwell, 1999; Ohh, 1999; Semenza, 2000). In VHL-defectivetumors, curiously enough, the two fundamental stages of tumordevelopment occur either simultaneously or in reverse, first triggeringthe hypoxia-cellular response, followed by proliferation of transformedcells, consistent with the angiogenic phenotype of tumors seen in theVHL syndrome (Ivanov, 2001). RCC is one of the few tumors wherespontaneous regression of metastatic disease has been documented aftertumor nephrectomy, treatment with placebo in phase III trials or afterinflammatory or infectious events (Bleumer, 2003; Michael, 2003). Theseobservations have provided strong evidence of the importance of theimmune system in the control of this cancer. Therefore, much attentionhas been focused on immunotherapeutic modalities for the treatment ofRCC.

SUMMARY OF THE INVENTION

Provided herein are monoclonal antibodies which bind immunospecificallyto the carbonic anhydrase IX (G250) protein. Specifically, such MAbsbind to the CA domain of the CA IX protein.

In one aspect, the invention provides an isolated antibody thatimmunospecifically binds to a carbonic anhydrase IX (G250) protein. Theantibody binds to the carbonic anhydrase (CA) domain of carbonicanhydrase IX (G250). Optionally, the monoclonal antibody reducescarbonic anhydrase activity when contacted with CA IX. Exemplaryantibodies of the invention include antibodies having a heavy chain witha CDR containing amino acids 99 to 111 of SEQ ID NO: 1 and a light chainwith a CDR containing amino acids 91 to 102 of SEQ ID NO: 2.Alternatively, the antibody has a heavy chain with a CDR1 containing theamino sequence SYAMS (SEQ ID NO: 55), XYAMX (SEQ ID NO: 56), or SYXMX(SEQ ID NO: 57). The antibody has a heavy chain with a CDR2 containingan amino sequence AISXXGGXTXXADSVKG (SEQ ID NO: 58) or AISGSGGSTTTADSVKG(SEQ ID NO: 59), or the antibody has a heavy chain with a CDR3containing an amino sequence NGNYRGSLXAFDI (SEQ ID NO: 60). The antibodyhas a light chain with a CDR1 containing an amino sequenceTGSSSNIGAGYDVH (SEQ ID NO: 91), or the antibody has a light chain with aCDR2 containing an amino sequence GNNNRPS (SEQ ID NO: 102), or theantibody has a light chain with a CDR3 containing an amino sequenceQSYDSSLSAWVV (SEQ ID NO: 63). The antibody is a monoclonal antibody oran scFv antibody or a minibody. Preferably, the binding affinity is fromabout 10⁻⁶ M to about 10⁻¹² M. Exemplary scFv antibodies include scFvantibodies having a sequence of SEQ ID NO: 3-44.

In another aspect, the invention provides an antibody complex containinga first fully human monoclonal antibody that binds to a carbonicanhydrase IX (G250) protein, operably linked to a second fully humanmonoclonal antibody that binds to a carbonic anhydrase IX (G250)protein. Generally, the antibody complex reduces carbonic anhydraseactivity. Preferably, the first and second antibodies do not bind to thesame epitope.

In a further aspect, the invention provides a nucleic acid sequencecontaining a first nucleic acid encoding an anti-CA IX antibody or ananti-CA IX scFv antibody and second nucleic acid encoding a cytokine,such as IL-2 or the T-cell receptor or portion or subunit thereof. Forexample, the second nucleic acid is the zeta chain of the T-cellreceptor complex. Optionally, the nucleic acid sequence contains a thirdnucleic acid sequence encoding a signaling region from a costimulatoryprotein such as CD28.

Also provided are methods for preventing or treating cancer byadministering to a person at risk or suffering from cancer atherapeutically effective amount of a first antibody that is an anti-CAIX antibody. The cancer is, e.g., renal cancer such as renal clear cellcancer. In certain embodiments, the method also includes administering asecond antibody that does not bind to the same epitope as the firstanti-CA IX antibody. The second antibody may bind to a CA IX protein oranother, non-CA IX protein. Exemplary antibodies that bind to non-CA IXproteins include Avastin, Erbitux, Humira, Xolair, Zavalin, Campath,Mylotarg, Herceptin, Remicaide, Simulect, Synagis, Zenapax, Rittman,Panorex, ReoPro, Oncoscint, and OKT3. Optionally, the method alsoincludes further administering a small molecule such as a neoplasticagent, or a cytokine, such as IL-2, GM-CSF, IL-12, or TNF-alpha.

The present invention also provides fusion proteins containing theantibodies of the invention. A fusion protein is, for example, ananti-CA XI antibody or a functional fragment thereof, operably linked toa cytokine or growth factor, such as an IL-2 polypeptide. Alternativelythe fusion protein contains an anti-CA XI antibody or a functionalfragment thereof, operably linked to an IgG molecule. In a furtherembodiment the fusion protein contains an anti-CA XI antibody or afunctional fragment thereof, operably linked to the T-cell receptor orfragment or subunit thereof. For example, the fusion protein containsthe zeta chain of the T-cell receptor compleX. Optionally, the fusionprotein contains a third polypeptide which includes a signaling regionfrom a costimulatory protein such as CD28.

Preferably, the antibody is human (i.e., the antibody does not containany non-human antibody proteins) or humanized (i.e., the antibody is anon-human antibody such as a mouse antibody that has been modified toremove the majority of their mouse protein sequences; generally only theantigen-recognized sites, or complementarily-determining hypervariableregions (CDRs) are of non-human origin). As used herein, a minibodycontains an antigen binding domain of an antibody and an immunoglobulinCH3 domain. For example, a minibody contains the binding domain of ananti-CA IX monoclonal antibody or scFv antibody, or a functionalfragment thereof, and a constant region of an immunoglobulin or fragmentthereof.

The invention also provides a composition containing an anti-CA IXmonoclonal antibody or scFv and a carrier. Optionally, the compositioncontains one or more other components, such as a cytokine (e.g., IL-2)or a small molecule, such as an antineoplastic agent. The invention alsoprovides a kit comprising, in one or more containers, thesecompositions.

In another aspect, the invention provides a method of quantitating theexpression of a protein on or in a mammalian cell by providing amammalian cell suspected of expressing a carbonic anhydrase IX (G250)protein, then contacting the cell with an anti-CA IX antibody or scFvantibody under conditions where the carbonic anhydrase IX (G250) proteinand the antibody are capable of forming a complex, and detecting theamount of complex formation.

The invention also provides a method of modifying an immune effectorcell by providing an immune effector cell obtained from a mammaliansubject having renal cancer and contacting the immune effector cell witha nucleic acid encoding an anti-CA IX antibody or scFv, or a fragmentthereof. The immune effector cell is a T cell. Generally, the nucleicacid comprises a vector, such as a retroviral vector. The invention alsoincludes the modified immune effector cell educated by these methods,and the use of these modified immune effector cells to in methods ofpreventing or treating cancer, by administering to a person at risk orsuffering from cancer (e.g. renal cancer) one or more modified immuneeffector cells. In such methods, one may also administer othercompounds, such as anti-neoplastic agents, or cytokines such as IL-2,GM-CSF, IL-12, and TNF-alpha.

The invention also provides a method of detecting a carbonic anhydraseIX (G250) protein by providing a first detection means that includes ananti-CA IX antibody or scFv that is operably linked to a support means,contacting this first detection means with a biological sample suspectedof containing a carbonic anhydrase IX (G250) protein under conditionswhere the carbonic anhydrase IX (G250) protein and the first detectionmeans are capable of forming a complex, and detecting the amount ofcomplex formation.

The invention further provides a method of detecting a carbonicanhydrase IX (G250) protein by contacting a biological sample suspectedof containing a carbonic anhydrase IX (G250) protein with a firstdetection means containing a first fully human monoclonal antibody thatbinds to the carbonic anhydrase (CA) domain of a carbonic anhydrase IX(G250) protein under conditions where the carbonic anhydrase IX (G250)protein and the first detection means are capable of forming a complex,contacting the biological sample with a second detection meanscontaining a second fully human monoclonal antibody that binds to thecarbonic anhydrase (CA) domain of a carbonic anhydrase IX (G250) proteinunder conditions where the carbonic anhydrase IX (G250) protein and thesecond detection means are capable of forming a complex, and detectingthe amount of complex formed between the carbonic anhydrase IX (G250)protein and the second detection means. In some embodiments, the firstand second antibodies do not bind to the same epitope. Optionally, thesecond detection means contains a detectable moiety.

In another aspect, the invention provides a non-invasive method ofdetecting a tumor in a subject by administering to the subject ananti-CA IX antibody or scFv antibody linked to a detectable moiety,allowing for the localization of the antibody in the tumor, anddetecting the detectable moiety. The detectable moiety comprises aradioactive element, and the detecting is performed by positron emissiontomography.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the carbonic anhydrase IX (G250)polypeptide. The signal peptide (SP), proteoglycan region (PG), carbonicanhydrase domain (CA), transmembrane domain (TM) and intracellulardomains (IC) are labeled. The amino acids corresponding to these regionsare provided below the regions.

FIG. 2 is a multiple sequence alignment of amino acid sequences ofsingle phage-scFv clones described herein. A consensus sequence isprovided in bold above the alignments. For example, antibody Clone A(36) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and a CDR3 with the amino acidsequence NGNYRGAFDI (SEQ ID NO: 65); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGAGFDVH (SEQ ID NO: 61); a CDR2with the amino acid sequence GNTNRPS (SEQ ID NO: 116); and a CDR3 withthe amino acid sequence QSYDSRLSAWV (SEQ ID NO: 108). Antibody Clone B(10) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and a CDR3 with the amino acidsequence NGNYRGAFDI (SEQ ID NO: 65); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 91); a CDR2with the amino acid sequence GNSNRPS (SEQ ID NO: 99); and a CDR3 withthe amino acid sequence QSYDRSLSWV (SEQ ID NO: 109). Antibody Clone C(119) has a heavy chain consisting of a CDR1 with the amino acidsequence SYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and a CDR3 with the amino acidsequence NGNYRGAFDI (SEQ ID NO: 65); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 91); a CDR2with the amino acid sequence GNTNRPS (SEQ ID NO: 116); and a CDR3 withthe amino acid sequence QSYDSTLRVWM (SEQ ID NO: 110). Antibody Clone D(6) has a heavy chain consisting of a CDR1 with the amino acid sequenceTYAMT (SEQ ID NO: 66); a CDR2 with the amino acid sequenceAVSGSGGSTYYADSVKG (SEQ ID NO: 72); and a CDR3 with the amino acidsequence GPVLRYGFDI (SEQ ID NO: 79); and a light chain consisting of aCDR1 with the amino acid sequence TGSRSNIGADYDVH (SEQ ID NO: 92); a CDR2with the amino acid sequence ANNNRPS (SEQ ID NO: 100); and a CDR3 withthe amino acid sequence QSYDSSLRAWV (SEQ ID NO: 111). Antibody Clone E(37) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and a CDR3 with the amino acidsequence NGNYRGAFDI (SEQ ID NO: 65); and a light chain consisting of aCDR1 with the amino acid sequence TGSRSNIGADYDVH (SEQ ID NO: 92); a CDR2with the amino acid sequence ANNNRPS (SEQ ID NO: 100); and a CDR3 withthe amino acid sequence QSYDSSLSAWV (SEQ ID NO: 112). Antibody Clone F(104) has a heavy chain consisting of a CDR1 with the amino acidsequence IYAMS (SEQ ID NO: 67); a CDR2 with the amino acid sequenceAISGSGGGTYHADSVKG (SEQ ID NO: 73); and a CDR3 with the amino acidsequence FSAYSGYDL (SEQ ID NO: 80); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGRGYNVH (SEQ ID NO: 93); a CDR2with the amino acid sequence DNTNRPS (SEQ ID NO: 101); and a CDR3 withthe amino acid sequence QSYDSGLRWV (SEQ ID NO: 113). Antibody Clone H(62) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and a CDR3 with the amino acidsequence NGNYRGAFDI (SEQ ID NO: 65); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 91); a CDR2with the amino acid sequence GNNNRPS (SEQ ID NO: 102); and a CDR3 withthe amino acid sequence QSYDKSLTWV (SEQ ID NO: 114). Antibody Clone I(45) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and a CDR3 with the amino acidsequence NGNYRGAFDI (SEQ ID NO: 65); and a light chain consisting of aCDR1 with the amino acid sequence TGTSSNIGAGYDVH (SEQ ID NO: 94); a CDR2with the amino acid sequence GNNNRPS (SEQ ID NO: 102); and a CDR3 withthe amino acid sequence QSYDKSLSWV (SEQ ID NO: 115). Antibody Clone K(106) has a heavy chain consisting of a CDR1 with the amino acidsequence SYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and a CDR3 with the amino acidsequence NGNYRGAFDI (SEQ ID NO: 65); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGAGFDVH (SEQ ID NO: 61); a CDR2with the amino acid sequence GNNNRPS (SEQ ID NO: 102); and a CDR3 withthe amino acid sequence QSYDSSLSAWV (SEQ ID NO: 116). Antibody Clone L(18) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO: 74); and a CDR3 with the amino acidsequence AAAGFDY (SEQ ID NO: 81); and a light chain consisting of a CDR1with the amino acid sequence TGSSSNIGRGYNVH (SEQ ID NO: 93); a CDR2 withthe amino acid sequence DDTNRPS (SEQ ID NO: 103); and a CDR3 with theamino acid sequence QSYDSSLRAWV (SEQ ID NO: 111). Antibody Clone M (39)has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO: 74); and a CDR3 with the amino acidsequence IGRYSSSLGY (SEQ ID NO: 82); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGRGYNVH (SEQ ID NO: 93); a CDR2with the amino acid sequence DNTNRPS (SEQ ID NO: 101); and a CDR3 withthe amino acid sequence QSYDSGLRWV (SEQ ID NO: 113). Antibody Clone N(94) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYGMH (SEQ ID NO: 68); a CDR2 with the amino acid sequenceVISYDGSNKYYADSVKG (SEQ ID NO: 75); and a CDR3 with the amino acidsequence EAPYSSSLDAFDI (SEQ ID NO: 83); and a light chain consisting ofa CDR1 with the amino acid sequence TGSSSNIGRGYNVH (SEQ ID NO: 93); aCDR2 with the amino acid sequence GNSNRPS (SEQ ID NO: 99); and a CDR3with the amino acid sequence HSRDNNGHHI (SEQ ID NO: 117). Antibody Clone0 (9) has a heavy chain consisting of a CDR1 with the amino acidsequence SYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO: 74); and a CDR3 with the amino acidsequence SHSSGGFDY (SEQ ID NO: 84); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGRGYNVH (SEQ ID NO: 93); a CDR2with the amino acid sequence GNTNRPS (SEQ ID NO: 116); and a CDR3 withthe amino acid sequence QSYDSSLSAWV (SEQ ID NO: 116). Antibody Clone P(21) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO: 74); and a CDR3 with the amino acidsequence SHSSGGFDY (SEQ ID NO: 84); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGRGYNVH (SEQ ID NO: 93); a CDR2with the amino acid sequence GNTNRPS (SEQ ID NO: 116); and a CDR3 withthe amino acid sequence QSYDSSLSAWV (SEQ ID NO: 116). Antibody Clone Q(27) has a heavy chain consisting of a CDR1 with the amino acid sequenceNYAMT (SEQ ID NO: 69); a CDR2 with the amino acid sequenceLISYDGSVTHYTDSVKG (SEQ ID NO: 76); and a CDR3 with the amino acidsequence GSGYQEH (SEQ ID NO: 85); and a light chain consisting of a CDR1with the amino acid sequence GGNNIGSKSVH (SEQ ID NO: 95); a CDR2 withthe amino acid sequence YDSDRPS (SEQ ID NO: 104); and a CDR3 with theamino acid sequence QVWDSSSDHHVV (SEQ ID NO: 118). Antibody Clone R(140) has a heavy chain consisting of a CDR1 with the amino acidsequence SYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO: 74); and a CDR3 with the amino acidsequence YGDYGSLDY (SEQ ID NO: 86); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 91); a CDR2with the amino acid sequence ANNNRPS (SEQ ID NO: 100); and a CDR3 withthe amino acid sequence QSYDSSLRAWV (SEQ ID NO: 111). Antibody Clone S(57) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISGSGVSTYYADSVKG (SEQ ID NO: 77); and a CDR3 with the amino acidsequence YCSSTSCYRGMDV (SEQ ID NO: 87); and a light chain consisting ofa CDR1 with the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 91); aCDR2 with the amino acid sequence ANNNRPS (SEQ ID NO: 100); and a CDR3with the amino acid sequence QSYDSSLRAWV (SEQ ID NO: 111). AntibodyClone T (82) has a heavy chain consisting of a CDR1 with the amino acidsequence SYGMH (SEQ ID NO: 68); a CDR2 with the amino acid sequenceVISYDGSNKYYADSVKG (SEQ ID NO: 75); and a CDR3 with the amino acidsequence GRAARPPFDY (SEQ ID NO: 88); and a light chain consisting of aCDR1 with the amino acid sequence SGSSSNIGSNYVY (SEQ ID NO: 96); a CDR2with the amino acid sequence RNNQRPS (SEQ ID NO: 105); and a CDR3 withthe amino acid sequence AAWDDSLNGVV (SEQ ID NO: 119). Antibody Clone V(98) has a heavy chain consisting of a CDR1 with the amino acid sequenceSYAMS (SEQ ID NO: 55); a CDR2 with the amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and a CDR3 with the amino acidsequence NGNYRGAFDI (SEQ ID NO: 65); and a light chain consisting of aCDR1 with the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 91); a CDR2with the amino acid sequence GNSNRPS (SEQ ID NO: 99); and a CDR3 withthe amino acid sequence QSYDSSLSAWV (SEQ ID NO: 116). Antibody Clone W(124) has a heavy chain consisting of a CDR1 with the amino acidsequence KYAMS (SEQ ID NO: 70); a CDR2 with the amino acid sequenceGISGSGGSTYYADSVKG (SEQ ID NO: 78); and a CDR3 with the amino acidsequence SSRSGYFLPLDY (SEQ ID NO: 89); and a light chain consisting of aCDR1 with the amino acid sequence QGNSLRYYYPS (SEQ ID NO: 97); a CDR2with the amino acid sequence GKNNRPS (SEQ ID NO: 106); and a CDR3 withthe amino acid sequence SSRDNTDNRVV (SEQ ID NO: 120). Antibody Clone X(125) has a heavy chain consisting of a CDR1 with the amino acidsequence SYGMH (SEQ ID NO: 68); a CDR2 with the amino acid sequenceAISGSGGSTYYADSVKG (SEQ ID NO: 74); and a CDR3 with the amino acidsequence AAVTGGFDP (SEQ ID NO: 90); and a light chain consisting of aCDR1 with the amino acid sequence GGDNIGRKSVH (SEQ ID NO: 98); a CDR2with the amino acid sequence DDRDRPS (SEQ ID NO: 107); and a CDR3 withthe amino acid sequence QVWDSSSKHYV (SEQ ID NO: 121).

FIG. 3 is a sequence alignment of human (HCA IX; SEQ ID NO:45) andmurine (MCA IX; SEQ ID NO: 46) CA IX amino acid sequence orthologs.Amino acids that are similar to or identical in human and murine CA IXpolypeptides are shaded.

FIG. 4 is a series of graphs showing FACS analyses of human RCC celllines (sk-rc-52, sk-rc-09, sk-rc-44, and sk-rc-59) contacted withpurified anti-CA IX scFv antibodies (G36, G37 and G119; Crt is acontrol).

FIG. 5 is a series of graphs showing FACS analyses of a human RCC cellline (sk-rc-52) contacted with increasing concentrations of purifiedanti-CA IX scFv antibodies (G10, G39, G92 and G98). The control graph isshown in the upper left.

FIG. 6 is a schematic illustration showing epitope mapping of theregions of the CA IX polypeptide to which various scFv antibodies of theinvention bind.

FIG. 7 is a schematic illustration showing methods for therapeuticantibody gene transfer.

FIG. 8 is a schematic illustration showing various methods fordiagnostic and therapeutic uses of the antibodies of the invention.

FIG. 9 is a schematic illustration showing a SIN lentiviral constructfor screening anti-CA IX scFvs.

FIG. 10 is a bar graph showing epitope mapping results demonstrating thebinding of various scFv antibodies to the CA domain of the CA IXprotein.

FIG. 11 is a schematic illustration of the construction of paramagneticproteoliposomes (PMPLs) containing CA IX (G250).

FIG. 12 is a photograph of a stained 12% SDS PAGE gel showingelectrophoresed G250 PMPLs.

FIG. 13 is a line graph showing saturation binding of anti-CA IX (G250)scFvs to stable G250-expressing 293T cells. The vertical axis is meanfluorescence intensity (MFI) and the horizontal axis is concentration ofthe antibody (in μg/ml).

FIG. 14 is a chart showing the quantitation of binding (IC₅₀) of anti-CAIX (G250) scFvs to stable G250-expressing 293T cells.

FIG. 15 is a photograph of a gel demonstrating amplification of a CA IX(G250) nucleic acid sequence from SK-RC renal cancer cell lines(sk-rc-52, -09, and -44). Beta actin is shown as a PCR control.

FIG. 16 is a line graph showing titration of anti-CA IX (G250) scFvs tostable G250-expressing 293T cells. The vertical axis is % of positivecells and the horizontal axis is concentration of the antibody (inμg/ml).

FIG. 17 is a series of graphs showing FACS analyses of a G250-positivehuman RCC cell line with anti-CA IX scFv-Fc fusion proteins. Comparedwith their monovalent counterparts, the divalent scFv-Fc fusion proteinsshowed more potent ability to bind to RCC cell line SK-RC-09 whichexpresses G250 molecule on the surface.

FIG. 18 is a series of graphs showing FACS analyses of a G250-negativehuman RCC cell line with anti-CA IX scFv-Fc fusion proteins.

FIG. 19 is a series of graphs showing FACS analyses of a G250-positivehuman RCC cell line with full lengths anti-CA IX human IgG. Cellstaining result indicated that full length human IgG showed much betterbinding to RCC cell line SK-RC-09 which expresses G250 molecule on thesurface comparing with their scFvs counterparts. In each panel the uppernumber is the positive percentage and the lower number is MFI for eachsample

FIG. 20 is a photograph of SDS-PAGE gel of seventeen scFv-Fc antibodyproteins under reducing and non-reducing conditions.

FIG. 21 is a line graph showing titration of anti-CA IX (G250) scFvs tostable G250-expressing 293T cells. The vertical axis is meanfluorescence intensity (GMFI) and the horizontal axis is concentrationof the antibody (in μg/ml).

FIG. 22 is a chart showing the quantitation of binding (IC₅₀) of anti-CAIX (G250) scFvs to stable G250-expressing 293T cells.

FIG. 23 is a table showing the results of cross competition ofanti-G250-FCs.

FIG. 24 is a bar chart showing inhibition of carbonic anhydrase activitywith carbonic anhydrase scFV-Fc specific antibodies.

FIG. 25 is a bar chart showing inhibition of carbonic anhydrase activitywith carbonic anhydrase scFV-Fc specific antibodies.

FIG. 26 is a table showing expression of anti-G250scFv and C9 TCRs on293T cell. Human primary Tcells were transduced with a self-inactivatinglentiviral vector encoding clone G36 anti-CA IX(G250) chimeric T-cellreceptor in cassette one and IRES driven expression of ZsGreen incassette two. After two overnight transductions on consecutive days, thecells were stained for chimeric T-cell receptor expression using APClabeled Mab 1D4 which is directed against the C9 tag located immediatelyafter the scFv and before the CD28 ECD. Transduction of two differenthuman donors is shown.

FIG. 27 is a bar chart showing the results of a Cytotoxicity assay usingG36 scFv-chimeric T cell receptor transduced T cells. Cytotoxicity waspreformed by incubating control non-transduced human T-cells (nonTd) orG36 anti-CA IX (G250) chimeric receptor transduced T cells (Td) withCAIX negative cells (Left panel—SK-RC-59) or CAIX positive cells (rightpanel—SK-RC-52 cells) at different effector to target cell ratios. Ascan be seen, these is highly efficient killing of the CAIX+SK-RC-52cells by transduced G36 anti-CA IX chimeric receptor expressing cellsvery poor killing of SK-RC-59 cells that do not express CAIX. Also asseen on the right panel, although there is some non-specific killing ofthe SK-RC-52 cells by non-transduced cells, the killing is much higherby the transduced cells.

FIG. 28 is a series of graphs showing FACS analyses of transduced 293Tcells with six self-inactivating lentiviral vectors encoding differentanti-CA IX chimeric T cell receptors in the first cassette and GFP inthe second cassette. Chimeric T cell receptors were identified bystaining with Mab 1D4 against the C9 tag.

FIG. 29 is a schematic illustration of the insertion of C9 tag into thetransmembrane region of TCR.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based in part on the discovery of antibodies that bindimmunospecifically to carbonic anhydrase IX (CA IX).

A number of mAbs have been identified that react with surface antigenson RCC. These include mAbs that recognize differentiation andoverexpressed antigens as well as mAbs that identify RCC-associatedantigens not expressed in normal kidney (Michael, 2003; Yang, 2003). Ofthese, antibodies against the carbonic anhydrase IX (CA IX), epidermalgrowth factor receptor (EGFR) and VEGF are the most studied and haveshown the greatest promise for treatment of RCC. The gene for CA IX,also known as G250 and MN is located on chromosomes 9p12 to 13 andencodes a transmembrane protein that binds zinc and has CA activity(Zavada, 1997; Grabmaier, 2000). In HeLa cells derived from humancarcinoma of cervix uteri and in RCC cell lines, CA IX/G250/MN/ is foundboth at the plasma membrane and as a nuclear protein with apparentmolecular weights of 58 and 54 kDa. It is N-glycosylated, and in thenonreduced state it forms oligomers (Pastorekova, 1992). Sequenceanalysis of the predicted CA IX protein shows that it contains a signalpeptide (aa 1-37), an extracellular (EC) part (aa 38-414), a hydrophobictransmembrane region of 20 amino acids (aa 415-434) and a smallC-terminus cytoplasmic portion of 25 amino acids (aa 435-459) (FIG. 1).The human and murine CA IX amino acid sequences are shown in FIG. 3. Theextracellular portion is composed of two distinct domains. The regionbetween the signal peptide and the CA domain (aa 53-111) showssignificant homology (38% identity) with a keratin sulfate attachmentdomain of a human large aggregating proteoglycan, aggrecan (Doege,1991). In the PG-like domain of CA IX, a hexapeptide motif withconsensus E-E-D-L-P-E (SEQ ID NO: 64) is repeated 7 times. The carbonicanhydrase domain is located close to the plasma membrane (aa 135-391).The CA IX antigen appears at malignant transformation and stainspositive in about 95% of clear cell RCC specimens as well as in mostrenal cell metastases. Results of a recent investigation focused on thegenes involved in VHL-mediated carcinogenesis demonstrateddown-regulation of CA IX gene expression in RCC cell lines by wild-typeVHL transgenes (Ivanov, 1998). Conversely, in VHL-defective tumors, CAIX is overexpressed (Ivanov, 2001).

Epitopes expressed on the cell surface of tumor cells are superiortargets for humoral anti-cancer therapy since, unlike intracellularantigens, they are accessible to circulating antibodies in vivo. In thelast few years, human monoclonal antibodies (mAbs) have become a welltolerated treatment option in an increasing number of cancers. Theconcept of selective tumor targeting with antibodies is based on theavid interaction between the antibody and an antigen that is expressedon malignant cells, but not on normal tissues. Many mechanisms have beenproposed for the ability of antibodies against tumors to mediate theireffects in vivo. For example, engagement of the antibody Fc domain witheffector cell FcγRs leads to antibody-dependent cell-mediatedcytotoxicity (ADCC). Some (antagonist or inhibitory) antibodies canblock the signaling on tumor cells and in this way may actsynergistically with immune effector responses by rendering the tumorcells more susceptible to immune effector cell triggered apoptosis orlytic cell death (Baselga, 1998). Another way that antibodies can beutilized is through the construction and functional expression ofchimeric-immune receptors or “T-bodies” on T-lymphocytes otherwise knownas “designer T-cells”. The antigen binding domain of the chimericreceptor consists of a single-chain antibody (scFv) while theintracellular signaling domain is derived from the cytoplasmic part of amembrane-bound receptor that is capable of inducing cellular activation(e.g. the FcεRI receptor γ-chain or the CD3 ζ-chain (Maher, 2002;Pinthus, 2003)). T-lymphocytes grafted with a chimeric receptor have thecombined advantages of MHC-independence and antibody-based antigenbinding with efficient T-cell activation upon specific binding to thereceptor ligand. This activation results in the production and secretionof cytokines such as IL-2, interferon, GM-CSF and TNF-∝.Antigen-specific lysis of tumor cells both in vitro and in vivo havebeen reported. T-lymphocytes can be permanently grafted withantigen-specific chimeric receptors by retroviral transduction of vectorconstructs encoding the receptor molecule of choice (reviewed byRivière, 2004).

Identification and Characterization of scFvs and Monoclonal Antibodies

Unique anti-CA IX scFvs were identified by sequencing analysis ofindividual clones. The VH and VL sequences of these scFvs are shown inFIG. 2. The gene families for these scFvs were VH3 for heavy chains andVL1 or VL3 for light chains. As described herein, the CA IX (G250)antibodies are human antibodies having a high affinity, and aregenerally directed to the CA domain of the protein. The CA domaincomprises amino acids 145-391 of the human CA IX protein. The antibodyof the invention binds to 5, 10, 20, 50, 100 or more residues of the CAdomain.

FACS Analysis of Anti-CA IX scFv Antibodies.

FACS analysis of several CA IX antibodies showed the specificity ofthese single chain antibodies for antigens present on a plurality ofhuman RCC cell lines, as shown in FIG. 4. Titration analysis of severalsingle chain antibodies, shown in FIG. 5, was also performed.

Epitope Characterization.

Primary epitope mapping of several single chain antibodies to the CA IXprotein showed that numerous antibodies are directed to the CA domainrather than the immunodominant PC domain, as shown in FIG. 6.Specificity of binding to the CA domain for several scFvs isdemonstrated in FIG. 13.

Internalization Studies.

Table 1 show the results of internalization studies of anti-CA IXscFv-Fc antibody fusion proteins into stable G250-293T cells.

TABLE 1 After 1 hour After 1 hour at 37°: at 4°: Clone: % Positive MFI %Positive MFI G10-Fc 99.98 2239 99.84 2017 G17-Fc 99.93 1260 99.96 1360G36-Fc 99.95 2029 99.96 2058 G39-Fc 99.92 1865 99.97 1918 G119-Fc 99.952064 99.99 2285 MFI = mean fluorescence intensity.Antibodies

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically binds” or“immunoreacts with” is meant that the antibody reacts with one or moreantigenic determinants of the desired antigen and does not react withother polypeptides. Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and F_(ab) expressionlibraries.

A single chain Fv (“scFv”) polypeptide molecule is a covalently linkedV_(H)::V_(L) heterodimer, which can be expressed from a gene fusionincluding V_(H)- and V_(L)-encoding genes linked by a peptide-encodinglinker. (See Huston et al. (1988) Proc Nat Acad Sci USA85(16):5879-5883). A number of methods have been described to discernchemical structures for converting the naturally aggregated, butchemically separated, light and heavy polypeptide chains from anantibody V region into an scFv molecule, which will fold into a threedimensional structure substantially similar to the structure of anantigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405;and 4,946,778.

Very large naïve human scFv libraries have been and can be created tooffer a large source of rearranged antibody genes against a plethora oftarget molecules. Smaller libraries can be constructed from individualswith infectious diseases in order to isolate disease-specificantibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43(1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3175-79 (1992)).

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.

The term “antigen-binding site,” or “binding portion” refers to the partof the immunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.”CDRs for the VH and VL regions of the scFv antibodies are shown in FIG.2.

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, an scFv, or a T-cellreceptor. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. For example, antibodies maybe raised against N-terminal or C-terminal peptides of a polypeptide.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity. Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to a CA IX epitope when the equilibriumbinding constant (K_(d)) is ≦1 μM, preferably ≦100 nM, more preferably≦10 nM, and most preferably ≦100 pM to about 1 pM, as measured by assayssuch as radioligand binding assays or similar assays known to thoseskilled in the art.

An CA IX protein of the invention, or a derivative, fragment, analog,homolog or ortholog thereof, may be utilized as an immunogen in thegeneration of antibodies that immunospecifically bind these proteincomponents.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a human monoclonal antibodyhas the same specificity as a human monoclonal antibody of the inventionby ascertaining whether the former prevents the latter from binding tothe CA domain of CA IX. If the human monoclonal antibody being testedcompetes with the human monoclonal antibody of the invention, as shownby a decrease in binding by the human monoclonal antibody of theinvention, then it is likely that the two monoclonal antibodies bind tothe same, or to a closely related, epitope.

Another way to determine whether a human monoclonal antibody has thespecificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with the CAIX protein, with which it is normally reactive, and then add the humanmonoclonal antibody being tested to determine if the human monoclonalantibody being tested is inhibited in its ability to bind the CA domain.If the human monoclonal antibody being tested is inhibited then, in alllikelihood, it has the same, or functionally equivalent, epitopicspecificity as the monoclonal antibody of the invention. Screening ofhuman monoclonal antibodies of the invention, can be also carried out byutilizing CA IX and determining whether the test monoclonal antibody isable to neutralize CA IX.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof (See, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference).

Antibodies can be purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The term “monoclonal antibody” or “MAb” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103).Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Fully human antibodies are antibody molecules in which the entiresequence of both the light chain and the heavy chain, including theCDRs, arise from human genes. Such antibodies are termed “humanizedantibodies”, “human antibodies”, or “fully human antibodies” herein.Human monoclonal antibodies can be prepared by using trioma technique;the human B-cell hybridoma technique (see Kozbor, et al., 1983 ImmunolToday 4: 72); and the EBV hybridoma technique to produce humanmonoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies may be utilized and may be produced by using human hybridomas(see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries. (See Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv (scFv) molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method,which includes deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

One method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. This method includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen, and a correlative methodfor selecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

The antibody can be expressed by a vector containing a DNA segmentencoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofCA IX in a sample. The antibody can also be used to try to bind to anddisrupt a CA IX activity.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof F_(ab) expression libraries (see e.g., Huse, et al., 1989 Science246: 1275-1281) to allow rapid and effective identification ofmonoclonal F_(ab) fragments with the desired specificity for a proteinor derivatives, fragments, analogs or homologs thereof. Antibodyfragments that contain the idiotypes to a protein antigen may beproduced by techniques known in the art including, but not limited to:(i) an F_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively,an antibody can be engineered that has dual Fc regions and can therebyhave enhanced complement lysis and ADCC capabilities. (See Stevenson etal., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies or toother molecules of the invention. (See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987)). Preferred linkers aredescribed in the literature. (See, for example, Ramakrishnan, S. et al.,Cancer Res. 44:201-208 (1984) describing use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No.5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against CA IX (G250)

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

Antibodies directed against a CA IX (G250) protein (or a fragmentthereof) may be used in methods known within the art relating to thelocalization and/or quantitation of a CA IX protein (e.g., for use inmeasuring levels of the CA IX protein within appropriate physiologicalsamples, for use in diagnostic methods, for use in imaging the protein,and the like). In a given embodiment, antibodies specific to a CA IXprotein, or derivative, fragment, analog or homolog thereof, thatcontain the antibody derived antigen binding domain, are utilized aspharmacologically active compounds (referred to hereinafter as“Therapeutics”).

An antibody specific for a CA IX protein of the invention can be used toisolate a CA IX polypeptide by standard techniques, such asimmunoaffinity, chromatography or immunoprecipitation. Antibodiesdirected against a CA IX protein (or a fragment thereof) can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibodies of the invention, including polyclonal, monoclonal, humanizedand fully human antibodies, may used as therapeutic agents. Such agentswill generally be employed to treat or prevent cancer in a subject. Anantibody preparation, preferably one having high specificity and highaffinity for its target antigen, is administered to the subject and willgenerally have an effect due to its binding with the target.Administration of the antibody may abrogate or inhibit or interfere withan activity of the AC IX protein.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Antibodies specifically binding a CA IX protein or a fragment thereof ofthe invention, as well as other molecules identified by the screeningassays disclosed herein, can be administered for the treatment of canceror other proliferative disorders in the form of pharmaceuticalcompositions. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington: The Science And Practice OfPharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co.,Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities,Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa.,1994; and Peptide And Protein Drug Delivery (Advances In ParenteralSciences, Vol. 4), 1991, M. Dekker, New York.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. (See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Theformulation can also contain more than one active compound as necessaryfor the particular indication being treated, preferably those withcomplementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

An antibody according to the invention can be used as an agent fordetecting the presence of CA IX (or a protein or a protein fragmentthereof) in a sample. Preferably, the antibody contains a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., F_(ab), scFv, orF_((ab)2)) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the invention can beused to detect an analyte mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of an analyte proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations, and immunofluorescence. In vitro techniques fordetection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Pharmaceutical Compositions

The antibodies or agents of the invention (also referred to herein as“active compounds”), and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the antibody oragent and a pharmaceutically acceptable carrier. As used herein, theterm “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate or otherwise interfere with an CA IX activity. Alsoprovided are methods of identifying compounds useful to treat cancer.The invention also encompasses compounds identified using the screeningassays described herein.

For example, the invention provides assays for screening candidate ortest compounds which modulate the CA IX carbonic anhydrase activity. Thetest compounds of the invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. (See, e.g., Lam, 1997. Anticancer DrugDesign 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates an CA IXactivity.

In another embodiment, at least one CA IX protein is provided, which isexposed to at least one neutralizing monoclonal antibody. Formation ofan antibody-antigen complex is detected, and one or more candidatecompounds are introduced to the complex. If the antibody-antigen complexis disrupted following introduction of the one or more candidatecompounds, the candidate compounds is useful to treat cancer or aproliferative disease or disorder, particularly a renal proliferativedisorder.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

Those skilled in the art will recognize that, in any of the screeningmethods disclosed herein, the antibody may be a CA IX neutralizingantibody. Additionally, the antigen may be a CA IX protein, or a portionthereof (e.g., the CA domain).

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. In the case of cell-free assayscomprising the membrane-bound forms of the CA IX proteins, it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of the proteins are maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody or the antigen (e.g. the CA IX protein or the CA domainthereof) can be immobilized utilizing conjugation of biotin andstreptavidin. Biotinylated antibody or antigen molecules can be preparedfrom biotin-NHS (N-hydroxy-succinimide) using techniques well-knownwithin the art (e.g., biotinylation kit, Pierce Chemicals, Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, other antibodies reactive withthe antibody or antigen of interest, but which do not interfere with theformation of the antibody-antigen complex of interest, can bederivatized to the wells of the plate, and unbound antibody or antigentrapped in the wells by antibody conjugation. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using such otherantibodies reactive with the antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic Assays

Antibodies of the present invention can be detected by appropriateassays, e.g., conventional types of immunoassays. For example, asandwich assay can be performed in which a CA IX protein or fragmentthereof (e.g., the CA domain) is affixed to a solid phase. Incubation ismaintained for a sufficient period of time to allow the antibody in thesample to bind to the immobilized polypeptide on the solid phase. Afterthis first incubation, the solid phase is separated from the sample. Thesolid phase is washed to remove unbound materials and interferingsubstances such as non-specific proteins which may also be present inthe sample. The solid phase containing the antibody of interest bound tothe immobilized polypeptide is subsequently incubated with a second,labeled antibody or antibody bound to a coupling agent such as biotin oravidin. This second antibody may be another anti-CA IX antibody oranother antibody. Labels for antibodies are well-known in the art andinclude radionuclides, enzymes (e.g. maleate dehydrogenase, horseradishperoxidase, glucose oxidase, catalase), fluors (fluoresceinisothiocyanate, rhodamine, phycocyanin, fluorescamine), biotin, and thelike. The labeled antibodies are incubated with the solid and the labelbound to the solid phase is measured. These and other immunoassays canbe easily performed by those of ordinary skill in the art.

The anti-CA IX antibodies and scFv antibodies of the invention, whenjoined to a detectable moiety, provides a way for detecting “canceroustissue” or tissue subject to aberrant cell proliferation and thereforeat risk for cancer. In addition to tissue that becomes cancerous due toan in situ neoplasm, for example, the antibody-detectable moietyconjugates also provides a method of detecting cancerous metastatictissue present in distal organs and/or tissues. Thus such tissue may bedetected by contacting tissue suspected of being cancerous with theantibody-detectable moiety under appropriate conditions to cause thedetectable moiety to be detected in cancerous tissue, thereby detectingthe presence of cancerous tissue.

The detectable moieties can be conjugated directly to the antibodies orfragments, or indirectly by using, for example, a fluorescent secondaryantibody. Direct conjugation can be accomplished by standard chemicalcoupling of, for example, a fluorophore to the antibody or antibodyfragment, or through genetic engineering. Chimeras, or fusion proteinscan be constructed which contain an antibody or antibody fragmentcoupled to a fluorescent or bioluminescent protein. For example,Casadei, et al., describe a method of making a vector construct capableof expressing a fusion protein of aequorin and an antibody gene inmammalian cells.

As used herein, the term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject (such as a biopsy), as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect cancer, a cancer cell, or a cancer-associated cell (suchas a stromal cell associated with a tumor or cancer cell) in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of CA IX include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. Furthermore, in vivo techniques for detection of CAIX include introducing into a subject a labeled anti-CA IX antibody. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. In embodiments, the invention provides a non-invasive methodof detecting a tumor or cancer cell in a subject. The subject isadministered an antibody or scFv antibody of the invention, where theantibody is linked to a detectable moiety (i.e., any moiety capable ofbeing detected by, e.g., fluorescent, chemical, chemiluminescent,radioactive, or other means known in the art), the antibody is allowedto localize to the tumor then is detected by observation of thedetectable moiety.

Localization of the detectable moiety. In the case of “targeted”conjugates, that is, conjugates which contain a targeting moiety—amolecule or feature designed to localize the conjugate within a subjector animal at a particular site or sites, localization refers to a statewhen an equilibrium between bound, “localized”, and unbound, “free”entities within a subject has been essentially achieved. The rate atwhich such an equilibrium is achieved depends upon the route ofadministration. For example, a conjugate administered by intravenousinjection to localize thrombi may achieve localization, or accumulationat the thrombi, within minutes of injection. On the other hand, aconjugate administered orally to localize an infection in the intestinemay take hours to achieve localization. Alternatively, localization maysimply refer to the location of the entity within the subject or animalat selected time periods after the entity is administered. By way ofanother example, localization is achieved when an moiety becomesdistributed following administration.

In all of the above cases, a reasonable estimate of the time to achievelocalization may be made by one skilled in the art. Furthermore, thestate of localization as a function of time may be followed by imagingthe detectable moiety (e.g., a light-emitting conjugate) according tothe methods of the invention, such as with a photodetector device. The“photodetector device” used should have a high enough sensitivity toenable the imaging of faint light from within a mammal in a reasonableamount of time, and to use the signal from such a device to construct animage.

In cases where it is possible to use light-generating moieties which areextremely bright, and/or to detect light-generating fusion proteinslocalized near the surface of the subject or animal being imaged, a pairof “night-vision” goggles or a standard high-sensitivity video camera,such as a Silicon Intensified Tube (SIT) camera (e.g., from HammamatsuPhotonic Systems, Bridgewater, N.J.), may be used. More typically,however, a more sensitive method of light detection is required.

In extremely low light levels the photon flux per unit area becomes solow that the scene being imaged no longer appears continuous. Instead,it is represented by individual photons which are both temporally andspatially distinct form one another. Viewed on a monitor, such an imageappears as scintillating points of light, each representing a singledetected photon. By accumulating these detected photons in a digitalimage processor over time, an image can be acquired and constructed. Incontrast to conventional cameras where the signal at each image point isassigned an intensity value, in photon counting imaging the amplitude ofthe signal carries no significance. The objective is to simply detectthe presence of a signal (photon) and to count the occurrence of thesignal with respect to its position over time.

At least two types of photodetector devices, described below, can detectindividual photons and generate a signal which can be analyzed by animage processor. Reduced-Noise Photodetection devices achievesensitivity by reducing the background noise in the photon detector, asopposed to amplifying the photon signal. Noise is reduced primarily bycooling the detector array. The devices include charge coupled device(CCD) cameras referred to as “backthinned”, cooled CCD cameras. In themore sensitive instruments, the cooling is achieved using, for example,liquid nitrogen, which brings the temperature of the CCD array toapproximately −120° C. “Backthinned” refers to an ultra-thin backplatethat reduces the path length that a photon follows to be detected,thereby increasing the quantum efficiency. A particularly sensitivebackthinned cryogenic CCD camera is the “TECH 512”, a series 200 cameraavailable from Photometrics, Ltd. (Tucson, Ariz.).

“Photon amplification devices” amplify photons before they hit thedetection screen. This class includes CCD cameras with intensifiers,such as microchannel intensifiers. A microchannel intensifier typicallycontains a metal array of channels perpendicular to and co-extensivewith the detection screen of the camera. The microchannel array isplaced between the sample, subject, or animal to be imaged, and thecamera. Most of the photons entering the channels of the array contact aside of a channel before exiting. A voltage applied across the arrayresults in the release of many electrons from each photon collision. Theelectrons from such a collision exit their channel of origin in a“shotgun” pattern, and are detected by the camera.

Even greater sensitivity can be achieved by placing intensifyingmicrochannel arrays in series, so that electrons generated in the firststage in turn result in an amplified signal of electrons at the secondstage. Increases in sensitivity, however, are achieved at the expense ofspatial resolution, which decreases with each additional stage ofamplification. An exemplary microchannel intensifier-based single-photondetection device is the C2400 series, available from Hamamatsu.

Image processors process signals generated by photodetector deviceswhich count photons in order to construct an image which can be, forexample, displayed on a monitor or printed on a video printer. Suchimage processors are typically sold as part of systems which include thesensitive photon-counting cameras described above, and accordingly, areavailable from the same sources. The image processors are usuallyconnected to a personal computer, such as an IBM-compatible PC or anApple Macintosh (Apple Computer, Cupertino, Calif.), which may or maynot be included as part of a purchased imaging system. Once the imagesare in the form of digital files, they can be manipulated by a varietyof image processing programs (such as “ADOBE PHOTOSHOP”, Adobe Systems,Adobe Systems, Mt. View, Calif.) and printed.

In one embodiment, the biological sample contains protein molecules fromthe test subject. One preferred biological sample is a peripheral bloodleukocyte sample isolated by conventional means from a subject.

The invention also encompasses kits for detecting the presence of CA IXor a CA IX-expressing cell in a biological sample. For example, the kitcan comprise: a labeled compound or agent capable of detecting a canceror tumor cell (e.g., an anti-CA IX scFv or monoclonal antibody) in abiological sample; means for determining the amount of CA IX in thesample; and means for comparing the amount of CA IX in the sample with astandard. The standard is, in some embodiments, a non-cancer cell orcell extract thereof. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect cancer in a sample.

Methods of Treatment

The invention provides for both prophylactic and therapeutic methods oftreating a subject at risk of (or susceptible to) a cancer, or othercell proliferation-related disease or disorder. Such diseases ordisorders include but are not limited to, e.g., those diseases ordisorders associated with aberrant expression of CA IX. Early symptomsof renal cancer include blood in the urine (hematuria), low back pain onone side, not associated with injury, a mass or lump in the abdomen,fatigue, weight loss that is not intentional, fever that is notassociated with a cold, flu, or other infection, and swelling of anklesand legs. Diagnosis of renal cancer may be performed by computedtomography scans, magnetic resonance imaging, intravenous pyelograms,ultrasonography and angiography.

Prophylactic Methods

In one aspect, the invention provides methods for preventing cancer or acell proliferative disease or disorder in a subject by administering tothe subject a monoclonal antibody or scFv antibody of the invention oran agent identified according to the methods of the invention. Forexample, a scFv or monoclonal antibody may be administered intherapeutically effective amounts.

Subjects at risk for cancer or cell proliferation-related diseases ordisorders include patients who have a family history of cancer or asubject exposed to a known or suspected cancer-causing agent.Administration of a prophylactic agent can occur prior to themanifestation of cancer such that the disease is prevented or,alternatively, delayed in its progression.

The appropriate agent can be determined based on screening assaysdescribed herein. Alternatively, or in addition, the agent to beadministered is a scFv or monoclonal antibody that prevents or inhibitscancer that has been identified according to the methods of theinvention.

Therapeutic Methods

Another aspect of the invention pertains to methods of treating a canceror cell proliferation-related disease or disorder in a patient. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein and/or an scFv antibodyor monoclonal antibody identified according to the methods of theinvention), or combination of agents that inhibit an activity of CA IX.

Combinatory Methods

The invention provides treating cancer in a patient by administering twoantibodies that bind to the same epitope of the CA IX protein or,alternatively, two different epitopes of the CA IX protein. Also, thecancer is treated by administering a first antibody that binds to CA IXand a second antibody that binds to a protein other than CA IX. Forexample, the second antibody is Avastin, Erbitux, Humira, Xolair,Zavalin, Campath, Mylotarg, Herceptin, Remicaide, Simulect, Synagis,Zenapax, Rittman, Panorex, ReoPro, Oncoscint, or OKT3.

Additionally, the invention provides administration of an antibody thatbinds to the CA IX protein and an anti-neoplastic agent, such a smallmolecule, a growth factor, a cytokine or other therapeutics includingbiomolecules such as peptides, peptidomimetics, peptoids,polynucleotides, lipid-derived mediators, small biogenic amines,hormones, neuropeptides, and proteases. Small molecules include, but arenot limited to, inorganic molecules and small organic molecules.Suitable growth factors or cytokines include an IL-2, GM-CSF, IL-12, andTNF-alpha. Small molecule libraries are known in the art. (See, Lam,Anticancer Drug Des., 12:145, 1997.)

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Construction and Characterization of a 27 BillionMember Non-Immune Human Single Chain Antibody (scFv) Phage DisplayLibraries

Two non-immune human scFv-phage display libraries containing 12 (MehtaI) and 15 (Mehta II) billion members were constructed that are used todirectly isolate a broad range of high affinity human scFvs against anytarget protein of interest. For details of this library and a list ofthe human antibodies that have been isolated from this library, pleasesee the National Foundation for Cancer Research (NFCR) website. ThesescFv libraries represents two of the largest human sFv-phage displaylibraries ever made (Nissim, 1994; Griffiths, 1994; Vaughan, 1996;Sheets, 1998, de Haard, 1999). For the studies described herein, theMehta I and II libraries are combined to streamline the panning andselection processes. Antibody libraries of this size are reliably beused to isolate high affinity antibodies to multiple epitopes on thetarget proteins. Two rounds of panning were performed against CAIX-PMPLS with 27 billion member human scFv phage display library. Fromeach round we picked out some clones and did small scale phage rescue.With those phage scFv antibody, ELISA based on both 293T-G250 cells andparental 293Tcells (No G250 expression) was performed. The clones whichonly bind to G250 positive cells were identified as “specific positive”as shown in above table and forwarded into further analysis. Results ofthe panning screen are provided below in Table E1.

TABLE E1 First panning Input Output Specific Positive 1st 5 × 1012 2.8 ×104 16/96 2nd 8 × 1012 7.6 × 106 109/192

Example 2 Construction of Prokaryotic and Eukaryotic CA IX ExpressionPlasmids

The CA IX cDNA gene was isolated from HeLa cells using RT-PCR and thencloned it into the pcDNA3.1 expression plasmid. CA IX is expressed inthree different forms for panning studies; 1) carboxy terminal C9-taggedfull-length protein for incorporation into paramagnetic proteoliposomesfor panning (Note: C9 is a 9 amino acid tag that corresponds to a regionof human rhodopsin); 2), carboxy-terminal C9-tagged extracellular domainof CA IX for expression of secreted protein for panning; and 3), fusionprotein between the extracellular domain of CA IX and Fc domain of IgG1.For epitope mapping studies, a construct was generated containing thewild-type GST-CA IX fusion protein (PG and CA extracellular domains),GST-PG domain and GST-CA domain and several GST fusion proteins withserial 5′ and 3′ truncations of CA.

Example 3 Isolation of High Affinity Human Anti-CA IX scFvs

Preparation of Paramagnetic Proteoliposomes Containing Properly Orientedand Functional CA IX and Panning—Paramagnetic proteoliposomes containingCA IX are prepared using recently reported procedures (Mirzabekov,2000). A schematic diagram outlining the procedure which we have used toprepare the paramagnetic proteoliposomes is shown in FIG. 14. Briefly,tosyl-activated Dynabeads are conjugated with 1D4 Mab and streptavidinand the non-covalently bound proteins are removed by washing. Theefficiency of antibody and streptavidin conjugation is checked by FACSusing PE-labeled anti-mouse IgG and FITC-biotin, respectively. COS-7cells are transiently or stably transfected with CA IX expressionplasmid that has been further engineered to contain a carboxy-terminalC9 tag to which the 1D4 mAb is directed. 24 hours later, cleared lysatesare obtained and are then incubated with a fixed ratio ofprotein/1D4-streptavidin-coated beads on a rocking platform. As a finalstep, after washing the beads are then mixed with solubilized lipidscontaining biotynil-DOPE, that self-assemble around the beads producingthe lipid bilayer. The inventors have previously obtained three highlypurified proteolipsomes containing human CCR5, human CXCR4 and EBV LMP1that are devoid of other cellular proteins. The orientation of CA IX ineach batch of CA IX-proteoliposomes is routinely confirmed by FACSanalysis using several well characterized M75 murine Mab against the ECdomain and a commercially available anti-CA IX Mab (Novus-Biologicals(NB 100-417).

The CA IX proteoliposomes are used to select recombinant antibodies.Briefly, after the final panning, as determined by fold enrichment intiters during each round of selection, a minimum of 200 individualcolonies (2 mictotiter plates) is infected with helper phage, and phagecontaining supernatants are screened using a cell based ELISA withstably transfected Cf2-CA IX+ cells (e.g., using a Tecan liquid handlingrobot so that thousands of colonies are evaluated). HRP-coupled rabbitanti-M13 phage is used for this purpose. Positive clones are rescreenedfor specificity by using Cf2 parental cell lines and several cell linesthat do not express CA IX. Initial screenings are made as stringent aspossible by lowering the number of cells used to coat the ELISA plate aswell as using different RCC cell lines that express different amounts ofsurface CA IX (e.g. SK-RC-52 (high expression); SK-RC-09 (lowexpression) or normal kidney cell lines (no expression)) (Ebert, 1990;Liu, 2002). Only phage that bind specifically to CA IX with values >5×background are further evaluated. DNA sequence analysis is performedusing a dedicated Perkin Elmer 310 Sequencer. Unique clones are enteredinto our scFv immunoglobulin gene database.

Example 4 Panning on Immunotube Coated Plates

Conventional panning procedures are used to isolate a panel of anti-CAIX scFvs by coating the purified recombinant CA IX C9 tagged protein (ECdomain) on immunotubes. The second procedure is used to decease anyepitope bias that may take place with the proteoliposome panning.Generally, the scFvs isolated from the two techniques are never thesame.

Example 5 Epitope Mapping of Anti-CA IX scFvs

Studies with GST-CA IX fusion proteins. Epitope mapping studies areperformed with GST-CA IX fusion proteins that are described herein.These studies are performed by coating ELISA plates with GST (control),GST-CA IX (wt), GST-CA IX (PG) or GST-CA truncation fusion proteins onthe plate and then performing a phage ELISA that detects phage antibodybinding by HRP-labelled rabbit anti-M13 antibody.Studies with CA IX point and deletion mutants—For other epitope mappingand binding affinity determination studies, the anti-CA IX scFvs areevaluated as highly purified soluble proteins that are produced in E.coli as scFv-His6-c-myc fusion proteins. Constructed are a series ofdeletion and alanine scanning mutations to the EC domain of C-terminalC9-tagged CA IX used to evaluate antibody binding through Co-IP/Westernblot studies. Briefly, eukaryotic expression plasmids for wt and mutantCA IX are transfected into 293T cells and the cell lysates are mixedwith the purified anti-CA IX scFvs and either IP'd with anti-C9 coatedsepharose beads (to assess CA IX expression levels) or with anti-c-mycsepharose (to assess co-IP with antibody) (see Sui, 2004). All of theanti-CA IX scFvs are tested for their ability to bind wt CA IX byWestern blotting under reducing, non-reducing conditions and afterPNGase F treatment to determine if the scFvs recognize linear orconformational epitopes or are sensitive to carbohydrate removal,respectively (Sui, 2004). The ability of the anti-CA IX scFvs to competewith WX-G250 and M75 by FACS analysis is also evaluated.Studies that examine cross-competition among different anti-CA IX scFvs.The mapping studies include cross-competition studies using thedifferent scFvs to determine if anti-CA IX scFvs that map to similarregions of CA IX are binding to the same epitope. For these studies, twodifferent approaches are used. In one approach the purified scFvproteins are biotinylated using standard techniques. In FACS assays,competitive binding (blocking) assays are used to determine the abilityof anti-CA IX scFvs to block the binding of any other biotinylatedscFvs, the later detected using streptavidin-FITC. In the secondapproach, real-time Biacore analysis is used to performmulti-determinant binding experiments. In these studies, when the firstscFv is saturably bound to the CA IX EC domain bound to the biosensorchip, a second scFv is added. If additional binding of the second scFvis detected, the epitopes are not overlapping. However, if additionalbinding of the second scFv is not detected, it can be assumed thateither steric hindrance or overlapping binding sites are responsible forthe lack of binding of the second scFv.

Example 6 Inhibition of Carbonic Anhydrase Activity Among DifferentAnti-CA IX scFvs

Anti-CA IX scFvs that are epitope mapped to the CA domain are able toblock enzymatic activity. This is an important anti-tumor biologicalproperty of the antibodies that is distinct from their retargetingcapacity. To evaluate the ability of anti-CA IX scFvs to block carbonicanhydrase activity, an assay is established to measure carbonicanhydrase activity of the CA IX paramagnetic proteoliposomes usingestablished methods (Brion, 1988; Dodgson, 1991). In brief, the velocityof the reaction CO₂+H₂O

H₂CO₃ is measured by the time required for acidification of carbonatebuffer in CO2 atmosphere, detected with phenol read as a pH indicator(Zavada, 1997). The reaction proceeds even in absence of the enzyme,with t_(o)=control time (this is set at 60 sec). Carbonic anhydrasereduces the time of acidification to t; one unit of the enzyme activityreduces the time to one half of control time: t/t_(o)=1. The CA IXparamagnetic proteoliposomes will be washed in PBS, resuspended in 1 mMcarbonic buffer, pH 8.0. Acetazolamide (Sigma) serves as a positivecontrol for inhibition of CA activity. Thus it is demonstrated that apurified anti-CA IX scFvs, in a dose dependent manner, blocks the CAactivity of the proteoliposomes.

Example 7 Affinity Measurements

Cell binding studies—Saturation binding studies on Cf2-CA IX+ cells areperformed on each of the purified anti-CA IX scFvs. The approximateaffinity of each scFv for CA IX is measured by serially diluting eachpurified anti-CA IX scFv prior to staining Cf2-CA IX+ cells. Cells areincubated with various concentrations of highly purified sFvs, washed,incubated with anti-c-myc, washed and then treated with FITC anti-mouseIgG. After final washing, cells are fixed and analyzed by FACS. Toaccount for variations in day-to-day staining and flow cytometercalibration, the EC₅₀ values for each scFv (defined as the concentrationof scFv which gives half-maximal MCF value) is normalized to thatobserved with WX-G250 in each experiment. Also examined is the abilityof the scFvs to bind to other CA IX expressing RCC cell lines thatexpress different levels and/or conformations of CA IX.BiaCore studies—The equilibrium dissociation constants (K_(D)) of theanti-CA IX scFvs are determined by surface plasmon resonance in aBIACORE 1000 instrument. The optimal conditions for immobilizing the CAIX EC domain to the biosensor chip is pre-determined. Association ratesare measured using a constant flow of 5 ul/min and a scFv concentrationsranging from 5×10⁻⁶ to 1×10⁻⁹M. k_(on) is determined from a plot of ln(dR/dt)/t vs concentration (Karlsson, 1991). Dissociation rates aremeasured using a constant flow of 25 ul/min and a sFv concentration of1.0×10⁻⁶M. k_(off) is determined during the first 30 seconds ofdissociation, K_(D) is calculated as K_(off)/K_(on) The bindingconstants of the monovalent scFvs are generally markedly improved whenthe binding sites are made bivalent due to an avidity effect. Therefore,cell binding and Biacore studies are performed on the bivalent scFv-Fcfusion proteins described herein.

Example 8 Antibody Induced CA IX Internalization

Anti-CA IX mediated internalization visualized by confocal microscopy.Crosslinking of membrane proteins is often a requirement for theirinternalization. Therefore the monovalent scFvs are converted tobivalent scFv-IgG1 fusion proteins. Constructed are eukaryoticexpression vectors that contain Sfi1/Not1 cloning sites that allow thescFvs identified from the phage display vector to be directly cloned inframe between a human VH leader sequence and Fc of human IgG1. Thisresults in the expression and secretion of bivalent scFv-IgG1 fusionproteins that is readily purified by protein A Sepharose. For thesestudies, each anti-CA IX scFv is cloned into the scFv-Fc expressionplasmid, transiently transfected into 293T cells and the secretedscFv-Fc fusion proteins are purified by protein A sepharose. The fusionproteins are directly labeled with FITC and their binding to RCC celllines SK-RC-52 (high CA IX expression); SK-RC-09 (low CA IX expression)or normal kidney cell lines (no expression)) are evaluated by con-focalmicroscopy. Cells are incubated with saturating amounts of FITC-anti-CAIX scFv-Fc at 4° C. or 37° C. for 60 minutes and then the cells aredirectly visualized for evidence of a change in staining from diffusesurface staining to one of capping and punctate staining of endocyticvesicles. Confocal images are recorded using an ACAS Ultima confocalmicroscope (Meridian Instruments, Inc.) with images representing 1-μmsections through the center of a focal plane using a 100× oil immersionobjective (Carnahan, 2003).

Example 9 Quantitation of CA IX Expression and Kinetics ofAntibody-Mediated CA IX Internalization for RCC Cell Lines

Cell surface expression of CA IX is quantitated using anti-CA IX scFv-Fcantibodies monvalently labeled with PE (conjugated by BD Biosources).One microgram of each scFv-Fc protein is generally added to cells withthe appropriate isotype controls for 30 min at 4° C. in the dark.Following washing and resuspension in FACS Lysing Solution (BDBiosciences), the cells are immediately analyzed by flow cytometry (BDFACSCalibur). The CA IX receptor numbers (determined by the binding ofat least three different scFv-Fc proteins) are calculated by comparisonto the standard fluorescence curve set by the QuantBRITE PE fluorescencequantitation kit (BD Biosciences) (Carnahan, 2003). Thecross-competition experiments described herein is a useful tool toquantitate antibody mediated internalization of CA IX. It is shown byFACS that adding increasing concentrations of one anti-CA IX scFv-Fcprotein does not compete for binding of a different PE-labelled anti-CAIX scFv-Fc protein under investigation. This provides a means ofindependently measuring cell surface CA IX and internalization. Toquantitate cell surface CA IX, SK-RC-52 or SK-RC-09 cells are incubatedwith increasing concentrations of unlabeled anti-CA IX scFv-Fc protein(range 0.01 to 100 μg/ml/10⁶ cells) for one hour at 37° C. Cells arethen washed with cold PBS and immediately analyzed by FACS for CA IXquantitation by addition of non-cross-competing PE-labelled anti-CA IXscFv-Fc protein. The resulting surface density of CA IX is measured bycomparing QuantiBRITE beads and PE-anti-CA IX scFv-Fc and are calculatedas a percentage of total CA IX on untreated cells (100%). The schemataare carried out for each anti-CA IX scFv-Fc for which non-cross reactiveantibodies have been identified. The kinetics of internalization areinvestigated by incubating saturating amounts of unlabeled scFv-Fc forvarying times prior to addition of PE-labelled anti-CA IX scFv-Fcprotein.

Example 10 Quantitation of CA IX Expression and Kinetics ofAntibody-Mediated CA IX Internalization for Primary RCC Cells

To determine if these observations can be extended to “fresh” renal cellcarcinoma cells, tumor tissue is obtained from human subjects who haveconsented to participate in DFHCC protocol 01-130: Collection ofspecimen in renal cell carcinoma. A pathologist determines that adequatematerial has been reserved for clinical diagnosis. Remaining tissue ismade available for research studies. A fragment of fresh tumor tissue iscollected into a sterile 50 cc conical tube containing tissue culturemedia. The specimen is delivered fresh on wet ice. The followingprotocol has been optimized by the cytogenetics laboratory within thePathology Department at Brigham & Women's Hospital. Cultures of primaryrenal tumors are routinely grown for clinical cytogenetics using thisprotocol. Cultures that have been successfully established are generallymaintained for eight passages or more using these methods. Specifically,non-malignant tissue is trimmed away from the specimen. The tumor isminced using sterile instruments. 4.5 mls of media are mixed with 0.5 mlof collagenase (Collagenase Type 1A (Sigma # C-9891)) solution. Thespecimen is incubated in collagenase for 48 hours at 37° C. The sampleis vigorously triturated to further disaggregate and transferred to a 15cc tube. The sample is centrifuged at 1000 RPM for 10 minutes, and thecell pellet resuspended in complete media (CM). Cells are plated incomplete media (CM) [DMEM medium supplemented with HEPES-buffer (10 mM),sodium pyruvate (1 mM), 20% (v/v) heat-inactivated FBS and penicillin(50 IU/ml)/streptomycin (50 μg/ml) onto tissue culture dishes or flasks.Cultures are grown in a tissue culture incubator at 37° C. with 10%humidified CO₂. Cultures are passaged and expanded when subconfluent.Aliquots are frozen in standard 90% FCS/10% DMSO freezing media after afew passages so that a bank of matched cells are available for futureexperiments. Quantitation of CA IX expression and kinetics ofantibody-mediated CA IX internalization are determined for freshlyprepared cells in the same manner as for the RCC cell lines.

Example 11 Evaluation of the Ability of Human T-Lymphocytes Transducedwith Lentiviral Vectors Encoding a Panel of Anti-CA IX Chimeric ImmuneReceptors to Kill CA IX Expressing RCC Cells In Vitro and In Vivo inSCID-Beige Mice Bearing RCC Xenografts

Lentiviral vectors have a distinct advantage over traditional retroviralvectors derived from oncogenic retroviruses (e.g. MMLV) in that they areable to efficiently transduce non-replicating cells. In addition, theVSV-G pseudotyped vectors can be concentrated to high levels (circa 10⁹infectious particles/ml) and by doing so high levels of transduction ofhuman peripheral blood T-lymphocytes are achieved. High level expansionsof these transduced cells are readily achieved. This provides a methodto simultaneously evaluate a number of different chimeric receptorsdirected against CA IX using single donor PBLs for transduction.

Design of Lentiviral Vectors. Previously constructed and characterizedare lentiviral vectors that are engineered with bicistronic expressioncassettes in which the first gene of interest (e.g. chimeric immunereceptor) is under the control on an internal CMV promoter and thesecond gene (eGFP or other) is expressed from an internal ribosomalentry site (EMCV). This configuration reliably leads to uniformexpression of both genes of interest (Ogueta, 2001; Zhu, 2004).Self-inactivating lentiviral vectors are used in these studies. Thelentiviral vectors encode different anti-CA IX chimeric immune receptorsand are transfected into 293T cells, along with a packaging vector andVSV-G envelope for production of virus particles (see Queta, 2002 andZhu, 2004). The viral supernatants are harvested, the virus particlesare then concentrated by ultracentrifugation and titers are determinedby FACS analysis of eGFP expression on freshly transduced 293T cells andPBMCs. Highly enriched transduced T-cells are readily selected for eGFP(or surface C9) expression by FACS sorting (see FIG. 5). As shown inFIG. 9, a C9 tag sequence is introduced to quantitate chimeric receptorexpression levels in the transduced cells.Isolation and transduction of human peripheral blood T-lymphocytes. Allblood samples are obtained either as buffy coat cells (isolated fromleukopacs (a leukophoresis product) through the Kraft Blood Bank at DFCIfrom anonymous donors) or from healthy volunteers after giving writteninformed consent under an institutional review board-approved protocol.PBLs are generally isolated by low-density centrifugation on Lymphoprep(Accurate Chemical and Scientific Corporation). For retroviraltransduction, T cells are activated overnight with 2 ug/mlphytohaemagglutinin (Murex Diagnostics) and transduced in 6-wellnon-tissue culture plates (Falcon) coated with 15 μg/ml of retronectin(Takara Biomedicals) as per the manufacturer's instructions, with freshviral supernatants daily at MOL 25 for 3 days by spinulation at 80 g atRT for 1 hr. The density of surface chimeric receptor expression isexamined by FACS analysis with PE-labeled 1D4 Mab (anti-C9 tag) and byWestern blotting of cell lysates.Design of Artificial Antigen Presenting Cells (AAPCs) and expansion oftransduced T-lymphocytes. To generate sufficient quantities of chimericreceptor expressing T-lymphocytes for in vivo studies, AAPCs areconstructed by retroviral transduction of NIH-3T3 mouse fibroblasts withCA IX and the co-stimulatory molecule CD80 (B7.1) molecule (3T3((CAIX+CD80+)). When the chimeric receptor expressing T-cells are incubatedwith AAPCs in the presence of IL-15, synergistically enhanced selectiveexpansion of the transduced cells is expected (Brentjens, 2003).Specifically, for ex vivo expansion, transduced T-lymphocytes areco-cultured in 6-well tissue culture plates (Falcon) with 80% confluentAAPSs in medium supplemented with 20 U/ml IL-2 and 10 ng/ml IL-15.Expansion of the transduced cells within the population of cells ismeasured by the progressive rise in percentage of cells expressing eGFPduring the culture period. In some studies, IL-2 production andfold-expansion of the transduced T-lymphocytes in the presence ofunmodified NIH3T3 cells or AAPCs are measured. Three days afterlentiviral transduction, the transduced cells are plated at 10⁶ PBLs/mlon the specified NIH3T3 monolayers. Supernatants are harvested after 24,48 and 72 hrs and assayed for IL-2 content by ELISA.Cytotoxicity assays. The cytotoxic activity of transduced T-lymphocytesis determined by standard ⁵¹Cr-release assays using RCC cell linesSK-RC-52 (high CA IX expression); SK-RC-09 (low CA IX expression) andnormal kidney cell lines (no CAIX expression) as target cells. Effectorcell number in all assays are calculated on the total number oftransduced T-lymphocytes (calculated by eGFP or C9 surface expression).Effector to target cell ratios are varied from 25:1 to 0.7:1. In somecases (primary RCC cells), CTL assays are performed using anonradioactive cytotoxicity detection kit (lactate dehydrogenase (LDH);Roche Diagnostics).

Example 12 Determination of Cytotoxicity of Anti-CA IX Antibodies InVivo

An RCC tumor model is established to test the killing capacity of thechimeric receptor expressing T-lymphocytes to kill RCC tumor cells invivo.

SCID-Beige mouse model of RCC—SCID-Beige mice (in groups of 5 mice) areinoculated with 5×10⁶ RCC positive and RCC negative tumor cell lines oneach flank. One week later, transduced T-lymphocytes (10×10⁶ cells or50×10⁶ cells) are injected by tail vein. In some animals, infusion ofIL-2 (Alzet pump) are used to augment in vivo cell activation at a rateof 5×10⁵ units/hr over 7 days. The tumor bearing mice are examined dailyfor signs of distress and tumor size are recorded with calipers. Two tothree different chimeric receptors are evaluated in vivo. Mocktransduced T-lymphocytes and T-lymphocytes transduced to express anirrelevant chimeric receptor serve as controls.

After the in vivo specificity of the anti-tumor effects are documented(no effects on CA IX negative tumor cell growth) the experiments arerepeated with only the CA IX+ RCC tumor cell inoculations. Mocktransduced T-lymphocytes and T-lymphocytes transduced to express anirrelevant chimeric receptor serve as controls. Survival curves areexamined for each group of mice. In separate tumor bearing mice,multiple weekly injections (up to three) of transduced cells are given.Statistical analysis of survival data log-rank analysis are performedusing GB-STAT softward (Dynamics Microsystems). Kaplan-Meier survivalcurves are plotted for all groups of mice.

Contribution of CD4+ and CD8+ transduced T-lymphocytes to in vivo tumorcell killing.

The role of CD4+ and CD8+ T-lymphocytes in tumor eradication is analyzedby treating the RCC-bearing mice with highly purified CD4+ and CD8+T-lymphocytes, or a 1:1 mixture of both. Survival curves are compared tomice inoculated with unfractionated transduced T-lymphocytes.

Survival of chimeric receptor expressing T-lymphocytes in vivo and tumorhistochemical staining. To determine the circulating half-life of thetransduced cells in vivo, mice are bled by tail vein at defined timesand the cells are analyzed for chimeric receptor expression by FACSanalysis. The number of cells and the density of the receptors as afunction of time are quantitated after infusion into the tumor-bearing(and as controls non-tumor bearing) mice. Some mice showing tumorregression are sacrificed and the tumor tissue are examined byimmunohistochemical staining for the presence of transduced cells.

Example 14 Analysis of Transduced Chimeric Receptor ExpressingT-Lymphocytes from RCC Patients for the Ability to Lyse Autologous TumorCells

It is recognized that long-term passage of cell lines in somecircumstances leads to genomic and gene expression changes that altertheir behavior as CTL targets. Primary RCC cells are used.Alternatively, passaged immortal RCC cell lines are used. In someembodiments, the primary RCC provide a target that most closelyresembles what T-lymphocytes generally encounter in vivo. Primary humanRCC cells maintained in short term culture have been used successfullyas targets of autologous CTLs (Liu, 1998; Kawai, 2003). Chimeric immunereceptors recognize antigens in a non-MHC-restricted manner soMHC-compatible tumor and T cells are not necessary. However, peripheralblood are collected from subjects donating tumor tissue so thatautologous lentiviral vector transduced T-lymphocytes are derived.

Transduction of RCC patient PBLs. Peripheral blood is generally obtainedunder standard protocols. Generally, cells are activated and transducedand optionally expanded as herein. Non-transduced and cells transducedwith an irrelevant chimeric receptor serve as controls since it ispossible that some degree of tumor cell killing are seen fromendogenously primed cytotoxic T-lymphocytes. Quantitation of thepercentage of transduced cells at the end of transduction and beginningof the cytoxicity assay are performed by FACS. Also quantitated is thedensity of chimeric receptors expressed on the transduced T-lymphocytes.Cells may be transduced once or a plurality of times (e.g., two, three,four, five or more times). One skilled in the art will recognize that incertain cases the level of chimeric receptor expression may be criticalfor cell killing. Therefore, the invention provides alternativepromoters that increase expression on transduced T-lymphocytes. Thedesign of the lentiviral vector system is flexible in the uniquerestriction sites flank the internal CMV promoter so that differentpromoters can be easily interchanged (e.g., SRα, EF1α, MMLV). Also,stable expression of a co-stimulator molecule (e.g. CD80 (B7.1)) isoptionally introduced.Cytotoxicity assay with autologous tumor cells. The surface density CAIX on the autologous RCC cells are quantitated by FACS analysis asdescribed herein. For the cytotoxicity assay, varying effector to tumorcell ratios are tested and ⁵¹Cr or LDH release are quantitated after a 4hr incubation.Examination of tumor cell killing by transduced T-lymphocytes.Visualization of cell killing in metastases is visualized with positronemission tomography. For example, in an RCC mouse model a MicroPETimaging system (Concorde Microsystems) is used. Generally, the mouse isinjected with about 200 μCi of ¹⁸F-fluorodeoxyglucose (FDG) injectedintravenously, and imaged.

Example 15 Analysis of Human Renal Cell Carcinomas

Human Peripheral Blood. Buffy coat cells obtained from leukopacs (aleukophoresis product) are obtained through the Kraft Blood Bank at DFCIfrom anonymous donors. Alternatively, human peripheral blood mononuclearcells from healthy volunteers are used.

Acquisition of Primary Human Tumor Specimens. Tumor tissues are obtainedfrom human subjects who have consented to participate in DFHCC protocol01-130. pathologist will determine that adequate material has beenreserved for clinical diagnosis. A fragment of fresh tumor tissue iscollected into a sterile 50 cc conical tube containing tissue culturemedia. The specimen is delivered fresh on wet ice to the researchlaboratory. Peripheral blood is also obtained from these patients. RCCtissue and blood is obtained from male and female subjects, and from aplurality of subjects representing diverse ethnicities.

Example 16 Non-Human Vertebrate Animals

SCID—Beige mouse model. 8 to 12-week old Fox Chase C.B.-17 (SCID-Beige)mice (Tanconic, Bermantown, N.Y.) are inoculated with tumor cells viasubcutaneous injection. Tumor cells (5×10⁶) are collected and loadedinto a needle (22-25 g) and injected subcutaneously above the hind flankregions creating a measurable induration. No suture is required. Noanesthesia is considered necessary. After 7 days, transduced cells(1-5×10⁷) are then injected by tail vein mixed in saline to a volume notexceeding 200 ul. All mouse studies are carried out under approvedprotocols. Administration of agents is accomplished by the use ofsubcutaneous pumps. Pumps implanted subcutaneously are placed betweenthe scapulae to minimize effect on mobility.

Example 17 Generation of Whole Human IgG1s: Fusion Proteins ContainingAnti-CA IX Antibodies or scFvs and Immunoglobulin Domains

Introduction of the anti-G250 scFv antibody G10 into TCAE6-LL2 wholeIgG1 expression vector was performed as follows. Primers used were

G10 VH 5′: (SEQ ID NO: 47) ATC GAC GCG TGC CTG AGC GAG GTG CAG CTG GTGCAG TC; G10 VH 3′: (SEQ ID NO: 48)CAA TGG TCA CCG TCT CTT CAG CTA GCA CCA GG; G10 VL5′: (SEQ ID NO: 49)ATC CCA AGC TTA AGC CAG TCT GTG CTG ACT CAG CC; G10 VL3′:(SEQ ID NO: 50) GGA GGG ACC AAA TTG ACC GTC CTA GGT CAG C.

VH and VL fragments of G10 were amplified by PCR with these primers andG10 scFv plasmid was used as template. The VH and VL PCR products weredigested by MluI/NheI and HindIII/AvrII, respectively, and inserted intocorresponding sites of full length human IgG1 expression vector. Aftertransformation and plasmid Maxi prep, sequence analysis was performed toconfirm that this clone is completed correctly. These methods are alsodescribed in Sui et al., PNAS 101(8): 2536-41 (2004), the contents ofwhich are incorporated by reference in their entirety.

Introduction of the anti-G250 scFv antibody G36 into TCAE6-LL2 wholeIgG1 expression vector was performed as described above with thefollowing primers.

G36 VH 5′: (SEQ ID NO: 51) TAG GGC ACG CGT GTG CTG AGC GAG GTG CAG CTGGTG CAG TC G36 VH 3′: (SEQ ID NO: 52)TCT AGT GCT AGC TGA AGA GAC GGT GAC CAT TG G36 VL5′: (SEQ ID NO: 53)CTA GCA AGC TTA TCC CAG TCT GTG CTG ACT CAG CC G36 VL3′: (SEQ ID NO: 54)ATA GCA CCT AGG ACG GTC AGC TTG GT

Example 18 Cross Competition of Anti-G250-Fcs for G250 Antigen Binding

Cross competition of anti-G250-Fcs for G250 Antigen binding wasperformed as follows:

Anti-G250-Fc antibody was labeled with Biotin (hot proteins) accordingto manufacturers instruction. 96 well microplates were coated withG250-Fc fusion proteins at 4° C. overnight, 50 μl ofbiotin-anti-G250-Fcs was added with/without cold anti-G250-Fcs at 5μg/ml in PBS, and incubate at room temperature for 1 hr wash out unboundantibodies and add HRP-strepdavidin. Plates were developed and OD450determinedCalculate %=100*(OD450 of hot protein plus cold protein)/(OD450 of hotprotein plus PBS)

Example 19 Inhibition of Carbonic Anhydrase Activity by CA IX SpecificscFv-Fc Antibodies

Inhibition of carbonic anhydrase activity by carbonic anhydrase specificscFv-Fc antibodies was determined as follows:

Material and Method:

The electrometric method to test the Carbonic Anhydrase (CA) activity inwhich the time required (in seconds) for a saturated CO2 solution tolower the pH of 0.012 M Tris HCl buffer from 8.3 to 6.3 at 0° C. isdetermined.

Blank Determination: Add 6.0 ml of chilled 0.02 M Tris HCl buffer, pH8.0 to a 50 ml Falcon tube. Maintain temperature at 0-4° C. and recordpH. Add 4 ml of chilled CO2 saturated water to Tris buffer andimmediately start a stopwatch to record the time required for the pH todrop from 8.3 to 6.3. Record this time as T₀.Enzyme Determination: Add 6.0 ml of chilled 0.02 M Tris HCl buffer, pH8.0 to a 50 ml Falcon tube. Maintain temperature at 0-4° C. and recordpH. Add 1 μg of Carbonic Anhydrase IX (CA IX), namely G250 (in theversion of extracellular domain of G250 fused to human IgG1 Fc domain,G250-ECD-Fc) in 100 μl of PBS. Quickly add 4 ml of CO2 saturated waterand record the time required for the pH to drop from 8.3 to 6.3. Recordthis time as T. Calculate the Unit activity of Carbonic Anhydrase as thefollowing formulation: Units/mg=2×(T₀−T)/(T×mg enzyme in reactionmixture).Inhibition Function of anti-G250 scFv-Fc antibodies determination: Mixanti-G250 scFv-Fc antibodies with 1 μg of G250-ECD-Fc at the molar ratioof Abs: Enzyme=1:1, 5:1 or 25:1 and incubated the mixture at roomtemperature for 50 minutes. Add 6.0 ml of chilled 0.02 M Tris HClbuffer, pH 8.0 to a 50 ml Falcon tube. Maintain the temperature at 0-4°C. and record pH. Add the mixture of antibody and G250-ECD-Fc, 4 ml ofCO2 saturated water and record the time required for the pH to drop from8.3 to 6.3. Record this time as T_(Ab). Carbonic anhydrase smallmolecular inhibitor acetazolamide (Sigma)) and anti-CXCR4 scFv-Fcantibody X33 are used as positive and negative control at the same molarratio, respectively. Calculate the Units activity of Carbonic Anhydrasetreated by scFv-Fc antibodies as the following formulation:Units_(Ab)/mg=2×(T₀−T_(Ab))/(T_(Ab)×mg enzyme in reaction mixture).Calculate the percentage of Inhibition as the following formulation: %of Inhibition=100×(1−Units_(Ab)/mg/Units/mg).

As shown in FIGS. 24 and 25, four of eighteen G250 specific scFv-Fcantibodies, which have been, tested show Carbonic Anhydrase inhibitionfunction at a dose-dependent pattern. The maximal inhibitions of cloneG6 and G39 reach about 50% while those for clone G37 and G125 are around40%. CA inhibitor acetazolamide almost abolishes the function ofG250-ECD-Fc at molar ratio of inhibitor:enzyme=1:1; on the other hand,the non-related antibody X33 doesn't have effect on the function of CA.

Example 20 PET Evaluation of RCC Metastases Using High-Affinity HumanAnti-CAIX Monoclonal Antibodies with Optimized PharmacokineticProperties

Renal cell carcinoma (RCC) accounts for 3% of all adult malignancies andthere are 36,000 new cases diagnosed each year in the United States. RCCis resistant to virtually all conventional modes of treatment, such asradiotherapy and chemotherapy. RCC is one of the few tumors wherespontaneous regression of metastatic disease has been documented aftertumor nephrectomy, treatment with placebo in phase III trials or afterinflammatory or infectious events. These observations have providedstrong evidence of the importance of the immune system in the control ofthis cancer. Therefore, much attention has been focused onimmunotherapeutic modalities for the treatment of RCC, including thetreatment with high-dose IL-2 which remains the preferred therapy forselect patients with metastatic RCC.

Carbonic anhydrase IX (CAIX) is a RCC-associated surface antigen that isnot expressed in normal kidney or other tissue except for epithelialcells of the bile ducts and small intestine and mucous cells of thegastric epithelium where in contrast to RCC, expression is localized tothe cytoplasm. CAIX (also known as G250 and MN) is a N-glycosylatedtransmembrane protein that binds zinc and has carbonic anhydrase (CA)activity). The extracellular portion is composed of two distinctdomains, a region between the signal peptide and the CA domain (aa53-111) shows significant homology with a keratin sulfate attachmentdomain of a human large aggregating proteoglycan, aggrecan and acarbonic anhydrase domain that is located close to the plasma membrane(aa 135-391). The CAIX antigen appears at malignant transformation andstains positive in about 95% of clear cell RCC specimens as well as inmost renal cell metastases. CAIX is thought to promote tumor cellproliferation in response to hypoxic conditions.

Several recent studies have focused on identifying molecular markersthat might predict the outcomes of patients with RCC.Immunohistochemical analysis of CAIX on paraffin embedded specimens from224 patients treated with nephrectomy for RCC. In this study, >90% oftumors expressed CAIX and its expression decreased with advancing stageof disease. Importantly, overall expression of CAIX was found todecrease with development of metastasis; as demonstrated by the lowerCAIX staining levels in metastatic lesions relative to matched primarytumor specimens. These findings were expanded by examining CAIXexpression in pathology specimens from 66 RCC patients who hadpreviously received IL-2 therapy. These results showed that high CAIXexpression was an important predictor of response to IL-2 therapy andprolonged survival. These and other studies suggest that improveddiagnostic strategies that incorporate CAIX expression may help toidentify those patients that are most likely to benefit from IL-2 basedtherapy which is associated with considerable toxicity and expense,making it an impractical standard therapy

Diagnostic and therapeutic Mabs against CAIX have been extensivelystudied by two groups in The Netherlands (murine G250 Mab and derivedcG250, chimeric Mab licensed to Wilex) and Czech Republic (murine MAbM75, licensed to Chiron). Both of these antibodies are directed againstthe PG domain of CAIX. Interestingly, attempts to produce Mabs againstother parts of CAIX through conventional immunization procedures in micehave been unsuccessful due to the immunodominance of the PG region andthe fact that this is the only region of CAIX that significantly differsbetween human and mouse homologues. The murine M75 Mab has been used inradioimmunoscintigraphy studies only in mice however, theseinvestigators have obtained similar pharmacokinetic, biodistribution andtumor localization results as have been reported in animal studies thathave used murine G250 Mab and the derived chimeric cG250 Mab. Thechimeric G250 mAb (WX-G250) is being developed by Wilex for bothdiagnostic and therapeutic purposes. The parental G250 and chimericcG250 Mabs have demonstrated excellent tumor targeting inimmunoscintigraphy studies in humans and limited clinical responses inradioimmunotherapy phase I trials of advanced RCC have been reported.Together, these studies clearly illustrate that CAIX is an excellenttumor-associated antigen for imaging of RCC in vivo and can provideimportant diagnostic and prognostic information that could markedlyimprove the clinical management of this disease.

MicroPET Evaluation of [I¹²⁴]-Labeled Antibody Fragments (scFvFc) inNon-Internalizing (CEA and CD20) Verses [Cu⁶⁴]-scFvFc Fragments forInternalizing (HER2 and PSCA) Tumor Antigen Systems.

The serum persistence of IgG1 and fragments with intact Fc region iscontrolled by the protective neonatal Fc receptor (FcRn) receptor.Essential for the FcRn binding, in both humans and rodents, are theresidues Ile253 and His310 in the CH2 domain and H435 in the CH3 domain(Kabat numbering system). MicroPET imaging has been used to examine thepharmacokinetics of several mutants of bivalent single-chain antibody(scFv)-Fc (dimer of Hinge-CH2-CH3 human IgG1, 105 kDa) fusion proteins(scFvFc) for their blood clearance properties and tumor imagingproperties. Specifically the double mutation H310A/H435Q (scFvFc DM) hasbeen shown to have superior tumor imaging in vivo. In particular, thefastest clearing scFvFc DM variant also exhibited high-contrast microPETimages in murine xenografts when labeled with [I¹²⁴] (t½=4.2 d) ascompared to wild-type and several single-mutant proteins (Kenanova,2005)

Production, Purification and In Vitro Characterization of HumanAnti-CAIX scFvFc DM Proteins.

scFvFc human IgG1 expression vectors have been constructed that containthe optimized H310A/H435Q double mutation as described above. Thiscassette accommodates all the human anti-CAIX scFvs described hereinthough unique in frame 5-Sfi1 and 3′-Not1 restriction sites after theIgG leader and before the doubly mutated Fc, respectively. Transienttransfection of 293F or 293FT cells using the calcium chlorideprecipitation technique results in high levels of antibody secretion,sufficient to obtain several milligrams of antibody from cells seeded on100 mm plates. Two or three of the highest affinity anti-CAIX scFvs thatmap to different regions (GP domain, carbonic anhydrase domain) will beused for production of scFvFc DM proteins.

Because the scFvFc DM proteins bind poorly to protein A (most likelybecause the FcRn binding region overlaps with protein A interactions athree-step purification scheme as described by Olafen, 2005 will beused. Briefly, culture supernatants will be dialyzed against 50 mmol/Lacetic acid (ph 5.0) before being loaded onto a cation exchange column(Poros HS20, Perkin-Elmer, Foster City, Calif.). Bound protein areeluted with a NaCl gradient and the eluted fractions, containing thescFvFc DM proteins are pooled and following buffer adjustments areloaded onto a hydroxyapatite column (Macro-Prep type I, Bio-Rad Labs).Bound proteins are eluted with a Kpi gradient and again the elutedfractions containing the scFvFc DM proteins will be pooled, bufferadjustments made and loaded onto an anion exchange column (Source 15Q,Amersham Biosciences Corp.). Bound proteins are eluted with a NaClgradient. The fractions with the scFvFc DM will be analyzed by SDS-PAGEand the fractions containing the purified antibodies will be pooled.Aliquots of purified proteins will be analyzed by SDS-PAGE underreducing and non-reducing conditions. Samples will also be subjected tosize-exclusion high-pressure liquid chromatography (HPLC) pm a Superdex200 HR 10/30 column (Amersham Biosciences). Retention times will becompared with standards. Binding of the purified proteins will beassessed by FACS analysis using CAIX(+)-SK-RC-52 and CAIX(−)-SK-59 humanRCC cells (obtained from Memorial Sloan-Kettering, NY)

Establishment of Orthotopic and Metastatic Models of RCC in AthymicMice.

Both spontaneous and experimental metastases model in female Ncr Nudemice (Taconic) will be established by intravenous and subcutaneousinjection of luciferase expressing CAIX(+)-SK-RC-52 and CAIX(−)-SK-59human RCC cells tumor cells, respectively. The presence of lung andother metastases will be assessed using the Xenogen imaging system. Forboth metastasis models, the experiments is expected to run forapproximately four to six weeks and each animal will be imaged weekly(or more frequently if required) by Xenogen imaging. For the intravenousadministration experiments, 0.3 ml of 10⁶ tumor cell suspension in PBSwill be injected into the tail vein of mice. Mice will be injected withD-Luciferin before performing the Xenogen imaging. At a time whenmetastases are present, the animals will be sacrificed and tissuescollected for histologically examination and immunohistology evaluationfor expression of CAIX and other HIF-inducible proteins including CXCR4,Glut-1 and other markers that may be of interest. For the subcutaneousadministration experiments, 0.3 ml of 10⁷ tumor cell suspension in PBSwill be orthotopically injected into mammary fat pads. Mice will bemonitored daily and when tumor diameter reach 1.5 cm, the primary tumorwill be surgically removed. These mice will also be subjected to Xenogenimaging and upon sacrifice tissues examined for metastases as describedabove.

Perform microPET Imaging on Athymic Mice Bearing Luciferase ExpressingG250(+)-SK-RC-52 Human Tumors Using Several Anti-G250 scFvFc DM Labeledwith [I¹²⁴] and [Cu⁶⁴].

In vivo pharmacokinetic and biodistribution studies will first becarried out in non-tumor bearing mice. Both [I¹²⁴] and [Cu⁶⁴] imagingwill be performed initially since it is not known at this time whetherCAIX undergoes efficient internalization either spontaneously or afterscFvFc DM antibody binding. As described in above [I¹²⁴] imaging isoptimal for non-internalizing receptors whereas [Cu⁶⁴] is very usefulfor imaging receptors that undergo internalization. Labeling with I¹²⁴(half-life, 4.2 d) is performed using the iodogen method (labile, goesonto tyrosines) whereas conjugation with DOTA is required to radiolabelwith Cu-64 (half-life, 12.7 h).

The ability to detect the tumors both with bioluminescence and microPETimaging will provide a powerful system to examine the sensitivity of theradiolabeled scFvFc DM proteins as imaging agents for metastaticlesions. MicroCT imaging will be used to provide anatomicallocalization.

Establishment of Stably Transfected CHO Cell Lines Secreting High Levelsof the Optimal Anti-CAIX scFvFc DM Proteins.

The imaging studies described above will provide important informationas to the lead anti-CAIX scFvFc DM protein that should be moved forwardfor human clinical studies. Specifically, human IgG1 expression plasmidsthat encode the dihydrofolate reductase (DHFR) gene, and the dominantselectable marker neomycin phosphotransferase (Neo) gene have beenconstructed. Very high levels of scFvFc DM protein production areinduced by forcing the antibody cassette to undergo gene amplificationby selection in methotrexate (MTX) for the dihydrofolate reductase gene.Amplification is achieved by increasing concentrations of MTX (5 nM→50nM→500 nM) to the CHO DG44 cells, the best amplificants from the 5 nMMTX stage are further amplified at the 50 nM and 500 nM stage. At thisstage, the selected amplificants are readapted to grow in spinnerflasks. During this time transfectoma antibody can be purified from thesupernatant. When the cell is producing 50-100 pg/cell/day and has adoubling time of 36 hrs or less, it will be considered a production cellline and a Parent Seed Stock will be prepared.

REFERENCES

-   Baselga J, Norton L, Albanell J, Kim Y M and Mendelsohn. Recombinant    humanized anti-HER2 antibody (Herceptin) enhances the anti-tumor    activity of paclitaxel and doxorubicin against HER2/neu    overexpressing human breast cancer xenografts. Cancer Res.    58:2825-2831, 1998.-   Brentjens R J, Latouche J-B, Santos E, Marti F, Gong M C, Lyddane C,    King P D, Larson S, Weiss M, Riviere I and Sadelain M. Eradication    of systemic B-cell tumors by genetically targeted huma T lymphocytes    co-stimulated by CD80 and interleukin-15. Nature Med. 9:279-286,    2003.-   Brion L P, Schwartz J H, Zavilowitz B J and Schwartz G J.    Micro-method for the measurement of carbonic anhydrase activity in    cellular homogenates. Anal. Biochem. 175:289-297, 1988.-   Carnahan J, Wang P, Kendall R, Chen C, Hu S, Boone T, Juan T,    Talvenheimo J, Montestruque S, Sun J, Elliott G, Thomas J, Ferbas J,    Kern B, Briddell R, Leonard JP and Cesano A. Epratuzumab, a    humanized monoclonal antibody targeting CD22: characterization of in    vitro properties. Blood 9:3982s-3900s (Suppl.), 2003.-   De Haard H J, Van Neer N, Reurs A, Hufton S E, Roovers R C,    Henderikx P, De Bruine A P, Arends J W, Hoogenboom HR. A large    non-immunized human fac fragment phage library that permits rapid    isolation and kinetic analysis of high affinity antibodies. J Biol    Chem 1999; 274(26):18218-18230.-   Dodgson S J, Tashian R E, Gross G and Carter N D. The carbonic    anhydrases. Plenum, New-York-London, 1991.-   Doege K J, Sasaki M, Kimura T and Yamada Y. Complete coding sequence    and deduced primary structure of the human cartilage aggregating    proteoglycan, aggrecan. J. Biol. Chem. 266:894-902, 1991.-   Ebert T, Bander N H, Finstad C L, Ramsawak R D and Old L J.    Establishment and characterization of human renal cancer and normal    kidney cell lines. Cancer Res. 50: 5531-5536, 1990.-   Guilford P. E-cadherin downregulation in cancer: fuel or fire? Mol.    Med. Today 5:172-177, 1999.-   Grabmaier K, Vissers J L M, DeWeijert M C A, Oosterwijk-Wakka J C,    van Bokhoven A, Bradenhiff R H, Noessner E, Mulders P A, Merkx G,    Figdor C G, Adema G J and Oosterwijk E. Molecular cloning and    immunogenicity of renal cell carcinoma-associated antigen G250.    Int. J. Cancer 85:865-870, 2000.-   Griffiths A D, Williams S C, Hartley O, Tomlinson I M, Waterhouse P,    Crosby W L, Kontermann R E, Jones P T, Low N M, Allison T J,    Prospero T D, Hoogenboom H R, Nissim A, Cox J P L, Harrison J L,    Zaccolo M, Gherardi E and Winter G. Isolation of high affinity human    antibodies directly from large synthetic repertoires. EMBO J 1994;    13: 3245-3260.-   Hanahan D. Folkman J. Paterns and emerging mechanisms of the    angiogenic switch during tumorigenesis. Cell 86:353-364, 1996.-   Hurwitz A A, Foster B A, Kwon E D, Truong T, Choi E M, Greenberg N    M, Burg M B and Allison J P. Combination immunotherapy of primary    prostate cancer in a transgenic mouse model using CTLA-4 blockade.    Cancer Res. 60:2444-2448, 2000.-   Ivanov S V, Kuzmin I, Wei M H, Pack S, Geil L, Johnson B E,    Stanbridge E J and Lerman M I. Down-regulation of transmembrane    carbonic anhydrases in renal cell carcinoma cell lines by wild-type    Hippel-Landau transgenes. Proc. Nat'l. Acad. Sci. 95:12596-12601,    1998.-   Ivanov S, Liao S-Y, Ivanova A, Danilkovitch-Miagkova A, Tarasova N,    Weirich G, Merrill M J, Proescholdt M A, Oldfield E H, Lee J, Zavada    J, Waheed A, Sly W, Lerman M I, and Stanbridge E J. Expression of    hypoxia-inducible cell-surface transmembrane carbonic anhydrase in    human cancer. Am. J. Pathol. 158:905-919, 2001.-   Karlsson R, Michaelsson A, and Mattson L. Kinetic analysis of    monoclonal antibody-antigen interactions with a new biosensor based    analytical system. J Immunol Methods 1991; 145:229-240.-   Kawai K, Saijo K, Oikawa T, Morishita Y, Noguchi M, Ohno T, Akaza H.    Clinical course and immune response of a renal cell carcinoma    patients to adaptive transfer of autologous cytotoxic T lymphocytes.    Clin. Ex. Immunol 134:264-269, 2003.-   Kwon E D, Foster B A, Hurwitz A A, Madias C, Allison J P, Greenberg    N M, and Burg M B. Elimination of residual metastatic prostate    cancer after surgery and adjunctive cytotoxic T    lymphocyte-associated antigen 4 (CTLA-4) blockade immunotherapy.    Proc. Nat'l. Acad. Sci. 96:15074-15079, 1999.-   Lamers C H J, Willemsen R A, Luider B A, Debets R, and Bolhuis R    L H. Protocol for gene transduction and expansion of human T    lymphocytes for clinical immunogene therapy of cancer. Cancer Gene    Ther. 9:613-623, 2002.-   Liao S Y, Brewer C, Zavada J, Pastorek J, Pastorekova S, Manetta A,    Bermann M L, DiSaia P J and Stanbridge E J. Identification of the MN    antigen as a diagnostic biomarker of cervical intraepithelial    squamous and glandular neoplasm and cervical carcinomas. Am. J.    Pathol. 145:598-609, 1994.-   Liu S Q, Kawai K, Shiraiwa H, Hayashi H, Akaza H, Hashizaki K, Shiba    R, Saijo K and Ohno T. High rate of induction of human autologous    cytotoxic T lymphocytes against renal carcinoma cells cultured with    an interleukin cocktail. Jpn. J. Cancer Res. 89:1195-1201, 1998.-   Liu Z, Smyth F E, Renner C, Lee F-T, Oosterwijk E and Scott A M.    Anti-renal cell carcinoma chimeric antibody G250: cytokine    enhancement of in vitro antibody-dependent cellular cytotoxicity.    Cancer Immunol. Immunother. 51:171-177, 2002.-   Maher J, Brentjens R J, Gunset G, Rivière I and Sadelain M. Human    T-lymphocyte cytotoxicity and proliferation directed by a single    chimeric TCRζ/CD28 receptor. Nature Biotech. 20:70-75, 2002.-   Maxwell P H, Wiesener M S, Chang G W, Clifford S C, Vaux E C,    Cockman M E, Wykoff C C, Pugh C S, Maher E R, and Ratcliffe P J. The    tumor suppressor protein VHL targets hypoxia-inducible factors for    oxygen-dependent proteolysis. Nature 399:271-275, 1999.-   Michael A and Pandha H S. Renal-cell carcinoma: tumour markers,    T-cell epitopes, and potential for new therapies. The Lancet Oncol.    4:215-223, 2003.-   Mirzabekov T, Kontos H, Farzan M, Marasco W, Sodroski J.    Paramagnetic Proteoliposomes Containing a Pure, Native, and Oriented    Seven-Transmembrane Segment Protein, CCR5. Nature Biotech. 2000;    18:649-654.-   Mostfi F K and Davis C J. WHO international histological    classification of tumors. Berlin: Springer, 1998.-   Nissim A, Hoogenboom H R, Tomlinson I M, Flynn G, Midgley C, Lane D    and Winter G. Antibody fragments from a ‘single pot’ phage display    library as immunochemical reagents. EMBO J 1994; 13:692-698.-   Ogueta S B, Yao F, and Marasco W A. Design and in vitro    characterization of a single regulatory module for efficient control    of gene expression in both plasmid DNA and a self-inactivating    lentiviral vector. Mol Med 2001; 7:569-71.-   Ohh M, Kaelin W G. The von Hipple-Lindau tumor suppressor protein:    New prospectives. Mol. Med. Today 6:257-263, 1999.-   Oosterwijk E, Ruiter D J, Hoedemaeker Ph J, PAuwels E K J, Jonas U,    Zwartendijk J and Warnaar S O. Monoclonal antibody G250 recognizes a    determinant present in renal-cell carcinoma and absent from normal    kidney. Int. J. Cancer 38:489-494, 1986.-   Parkkila S, Rajaniemi H, Parkkila A-K, Kivela J, Waheed A,    Pastorekova S, Pastorek J and Sly W S. Carbonic anhydrase inhibitor    suppresses invasion of renal cancer cells in vitro. Proc. Nat'l.    Acad. Sci. 97:2220-2224, 2000.-   Pastorek J, Pastorekova S, Callebaut I, Mornon J P, Zelnik V,    Opaysky R, Zatovicova M, Liao S, Portelle D, Stanbridge E J, Zavada    J, Burny A and Kettmann R. Cloning and characterization of MN, a    human tumor-associated protein with a domain homologous to carbonic    anhydrase and a putative helix-loop-helix DNA binding segment.    Oncogene 9:2788-2888, 1994.-   Pastoreková S, Závadová Z, Ko{hacek over (s)}t′ál M, Babu{hacek over    (s)}iková O and Závada J. A novel quasi-viral agent, MaTu, is a    two-component system. Virology 187:620-626, 1992.-   Pinthus J H, Waks T, Kaufman-Francis K, Schindler D G, Harmelin A,    Kanety H, Ramon J and Eshhar Z. Immuno-gene therapy of established    prostate tumors using chimeric receptor-redirected human    lymphocytes. Cancer Res. 63:2470-2476, 2003.-   Rivière I, Sadelain M and Brentjens R J. Novel strategies for cancer    therapy: The potential of genetically modified T lymphocytes. Curr.    Hematol. Reports. 3:290-297, 2004.-   Salmon P, Kindler V, Ducrey O, Chapuis B, Zubler R H, Trono D.    High-level transgene expression in human hematopoietic progenitors    and differentiated blood lineages after transduction with improved    lentiviral vectors. Blood 96:3392-3398, 2000.-   Semenza G L. Hypoxia, clonal selectin, and the role of HIF-1 in    tumor progression. Crit. Rev. Biochem. Mol. Biol. 35:71-103, 2000.-   Sheets M D, Amersdorfer P, Finnern R, Sargent P, Lindqvist E, Schier    R, Hemingsen G, Wong C, Gerhart J C, and Marks J D. Efficient    construction of a large nonimmune phage antibody library: The    production of high-affinity human single-chain antibodies to protein    antigens. Proc Natl Acad Sci USA 1998; 956:6157-6162.-   Steffens M G, Boeman O C, Oosterwijk-Wakka J C, Oosterhof G O N,    Witjes J A, Koenders E B, Oyen W J G, Buijs W C A M, Debruyne F M J,    Corstens F H M and Oosterwijk E. Targeting of renal cell carcinoma    with iodine-131-labeled chimeric monoclonal antibody G250. J. Clin.    Oncol. 15:1529-1537, 1997.-   Steffens M G, Boerman O C, de Mulder P H M, Oyen W J G, Buijs W C A    M, Witjes A, van den Broek W J M, Oosterwijk-Wakka J C, Debruyne F M    J, Corstens F H M, and Oosterwijk E. Phase I radioimmunotherapy of    metastatic renal cell carcinoma with ¹³¹I-labeled chimeric    monoclonal antibody G250. Clin. Cancer Res. 5:3268s-3274s, 1999.-   Sui J, Li W, Murakami A, Tamin A, Matthews L J, Wong S K, Moore M J,    Tallrico A S, Olurinde M, Choe H, Anderson L J, Bellini W J, Farzan    M, and Marasco W A. Potent Neutralization of SARS Coronavirus    Infection by a Human Monoclonal Antibody Against the ACE2-Binding    Domain of Spike Protein. Proc Natl Acad Sci USA. 2004 Feb. 24;    101(8):2536-41.-   Svastova E, Zilka N, Zatovicova M, Gibadulinova A, Ciampor F,    Pastorek J and Pastorekova S. Carbonic anhydrase IX reduces    E-cadherin-mediated adhesion of MDCK cells via interaction with    β-catenin. Exp. Cell Res. 290:332-345, 2003.-   Tsui L V, Kelly M, Zayek N, Rojas V, Ho K, Ge Y, Moskalenko M,    Mondesire J, Davis J, Van Roey M, Dull T and McArthur J G.    Production of human clotting factor IX without toxicity in mice    after vascular delivery of a lentiviral vector. Nature Biotech.    20:53-57, 2002.-   Vaughan T J, Williams A J, Pritchard K, Osbourn J K, Pope A R,    Earnshaw J C, McCafferty J, Hodits R A, Wilton J, and Johnson K S.    Human antibodies with sub-nanomolar affinities isolated from a large    non-immunized phage display library. Nature Biotech 1996; 14:309.-   Walsh P C, Retik A B, Vaughan E D, Wein A J, Kavoussi L R, Novick A    C, Partin A W, and Peters C A. Campbell's Urology 8^(th) edition.    Philadelphia, London, New York, St. Louis, Sydney, Toronto:    Saunders; 2003.-   Weijtens M E M, Willemsen R A, Valerio D, Stam K and Bolhuis R L H.    Single chain Ig/γ gene-redirected human T lymphocytes produce    cytokines, specifically lyse tumor cells, and recycle lytic    capacity. J. Immunol 157:836-843, 1996.-   Yang J C, Haworth L, Sherry R M, Hwu P, Schwartzentruber D J,    Topalian S L, Steinberg S M, Chen H X and Rosenberg S A. A    randomized trial of bevacizumab, an anti-vascular endothelial growth    factor antibody, for metastatic renal cancer. N. Eng'l. J. Med.    349:427-434, 2003.-   Zatovicova M, Tarabkova K, Svastova E, Gibadulinova A, Mucha V,    Jakubickova L, Biesova Z, Rafojova M, Gut M O, Parkkila S, Parkkila    A-K, Waheed A, Sly W S, Horak I, Pastorek J and Pastorekova S. J.    Immunol. Methods 282L117-134, 2003.-   Závada J, Závadová Z, Machon O, Kutinova L, Nemeckova S, Opaysky R    and Pastorek J. Transient transformation of mammalian cells by MN    protein, a tumor-associated cell adhesion molecule with carbonic    anhydrase activity. Int. J. Oncol. 10:857-863, 1997.-   Závada J, Závadová Z, Pastoreková S, Caimpor F, Pastorek J and    Zelnik V. Expression of MaTu-MN protein in human tumor cultures and    in clinical specimens. Int. J. Cancer 54:268-274, 1993.-   Závada J, Závadová Z, Pastorek J, Biesova Z, Jezek J and Velek J.    Human tumour-associated cell adhesion protein MN. CA IX:    identification of M75 epitope and of the region mediating cell    adhesion. Brit. J. Cancer 82(11):1808-1813, 2000.-   Zhu Q, Ricardo R R, Zhang L, Ogueta S B, Agrawal R S, Dzau V J, and    Marasco W A. Development of constitutive and inducible    self-inactivating lentiviral vectors and their application in    cardiovascular gene transfer. Gene Ther Mol Biol., 2004; (8):    91-102.-   Zufferey R, Dull T, Mandel R J, Bukovsky A, Quiroz D, Naldini L and    Trono D. Self-inactivating lentivirus vector for safe and efficient    in vivo gene delivery. J. Virol. 72:9873-9880, 1998.

OTHER EMBODIMENTS

Although particular embodiments have been disclosed herein in detail,this has been done by way of example for purposes of illustration only,and is not intended to be limiting with respect to the scope of theappended claims, which follow. In particular, it is contemplated by theinventors that various substitutions, alterations, and modifications maybe made to the invention without departing from the spirit and scope ofthe invention as defined by the claims. Other aspects, advantages, andmodifications considered to be within the scope of the following claims.The claims presented are representative of the inventions disclosedherein. Other, unclaimed inventions are also contemplated. Applicantsreserve the right to pursue such inventions in later claims.

We claim:
 1. A chimeric T cell receptor comprising an intracellularsignaling domain, a transmembrane domain and an extracellular domaincomprising a humanized carbonic anhydrase IX (G250)-antibody or fragmentthereof, wherein the carbonic anhydrase IX (G250) antibody has heavychain comprising a) a CDR1 comprising an amino acid sequence SYAMS (SEQID NO: 55); b) a CDR2 comprising an amino acid sequenceAISANGGTTYYADSVKG (SEQ ID NO: 71); and c) a CDR3 comprising an aminoacid sequence NGNYRGAFDI (SEQ ID NO: 65); and i) a light chain with aCDR1 comprising an amino sequence TGSSSNIGAGFDVH (SEQ ID NO: 61); a CDR2comprising an amino sequence GNTNRPS (SEQ ID NO: 116); and a CDR3comprising an amino sequence QSYDSRLSAWV (SEQ ID NO: 108).
 2. A chimericT cell receptor comprising an intracellular signaling domain, atransmembrane domain and an extracellular domain, the extracellulardomain comprising a humanized carbonic anhydrase IX (G250)-antibody orfragment thereof, wherein the carbonic anhydrase IX (G250) antibody is ascFv antibody and has a heavy chain comprising an amino acid sequence ofSEQ ID NO: 3 and wherein said scFv antibody has a light chain comprisingan amino acid sequence of SEQ ID NO: 24.